Glatiramer acetate-specific T cells in the brain
`express T helper 2兾3 cytokines and
`brain-derived neurotrophic factor insitu
`
`Rina Aharoni*, Basak Kayhan*, Raya Eilam†, Michael Sela*‡, and Ruth Arnon*
`
`Departments of *Immunology and †Veterans Resources, The Weizmann Institute, Rehovot 76100, Israel
`
`involved in the therapeutic effect induced by GA on MS as well
`(5–7). Furthermore, we have recently demonstrated that GA-
`specific regulatory cells, induced in the periphery either by
`injection or by oral treatment with GA, pass the blood–brain
`barrier and accumulate in the CNS. This finding was manifested
`by their isolation from brains of actively sensitized GA-treated
`mice and by localization of GA-specific cells in the brain after
`their passive transfer to the periphery (8, 9).
`Although the presence of GA-specific Th2 cells in the CNS
`was confirmed, their ability to actually secrete Th2 cytokines and
`induce therapeutic effect in situ has not been verified. Some
`reservation concerning the capability of T cells to secrete Th2
`cytokines in the CNS was raised after finding that the CNS
`environment induces bias toward the Th1 pathway and that
`myelin basic protein-specific Th2 cells can aggravate EAE (6,
`10). It was therefore essential to elucidate whether the GA-
`specific cells accumulating in the brain actually function as
`suppressor cells in the diseased organ and secrete antiinflam-
`matory cytokines in situ.
`It was recently demonstrated that GA-specific cells of both
`mouse and human origin also secrete the potent brain-derived
`neurotrophic factor (BDNF) (3, 11), which induces axonal
`outgrowth, remyelination, regeneration, and rescue of degener-
`ating neurons (12, 13). Yet, this BDNF secretion was found only
`in peripheral T cell lines in vitro and not in the target organ.
`BDNF expression by GA-specific cells in the CNS may indicate
`that GA activity is not restricted to antiinflammatory effect but
`generates actual neuroprotection and regeneration processes as
`well. In this study, we investigated whether GA-induced T cells
`that penetrate the brain do function as regulatory cells by
`producing Th2兾3 cytokines and neurotrophic factor. We wish to
`report that, indeed, the cytokines IL-10, transforming growth
`factor ␤(TGF-␤), and BDNF are expressed by GA-induced cells
`in situ.
`
`Materials and Methods
`Mice. (SJL兾JxBALB兾c)F1 female mice, 10–16 weeks old, were
`purchased from The Jackson Laboratory.
`
`Glatiramer Acetate. GA consists of acetate salts of synthetic
`polypeptides, containing four amino acids: L-alanine, L-
`glutamate, L-lysine, and L-tyrosine (4). GA from batch no.
`242990599, with an average molecular weight of 7,300, was
`obtained from Teva Pharmaceutical Industries (Petach Tikva,
`Israel) and used throughout the study.
`
`T Cells. GA-specific cells from two T cell lines established from
`spleens of mice that had received daily injections (2 mg兾day, 10
`
`IMMUNOLOGY
`
`Abbreviations: GA, glatiramer acetate; BDNF, brain-derived neurotrophic factor; TGF-␤,
`transforming growth factor ␤; MS, multiple sclerosis; EAE, experimental autoimmune
`encephalomyelitis; GFAP, glial fibrillary acidic protein; Th, T helper.
`‡To whom correspondence should be addressed. E-mail: michael.sela@weizmann.ac.il.
`
`© 2003 by The National Academy of Sciences of the USA
`
`Contributed by Michael Sela, September 25, 2003
`
`The ability of a remedy to modulate the pathological process in the
`target organ is crucial for its therapeutic activity. Glatiramer
`acetate (GA, Copaxone, Copolymer 1), a drug approved for the
`treatment of multiple sclerosis, induces regulatory T helper 2兾3
`cells that penetrate the CNS. Here we investigated whether these
`GA-specific T cells can function as suppressor cells with therapeutic
`potential in the target organ by in situ expression of T helper 2兾3
`cytokines and neurotrophic factors. GA-specific cells and their in
`situ expression were detected on the level of whole-brain tissue by
`using a two-stage double-labeling system: (i) labeling of the
`GA-specific T cells, followed by their adoptive transfer, and (ii)
`detection of the secreted factors in the brain by immunohistolog-
`ical methods. GA-specific T cells in the CNS demonstrated intense
`expression of the brain-derived neurotrophic factor and of two
`antiinflammatory cytokines, IL-10 and transforming growth factor
`␤. No expression of the inflammatory cytokine IFN-␥was observed.
`This pattern of expression was manifested in brains of normal and
`experimental autoimmune encephalomyelitis-induced mice to
`which GA-specific cells were adoptively transferred, but not in
`control mice. Furthermore, infiltration of GA-induced cells to the
`brain resulted in bystander expression of IL-10 and transforming
`growth factor ␤by resident astrocytes and microglia. The ability of
`infiltrating GA-specific cells to express antiinflammatory cytokines
`and neurotrophic factor in the organ in which the pathological
`processes occur correlates directly with the therapeutic activity
`of GA in experimental autoimmune encephalomyelitis兾multiple
`sclerosis.
`
`Neurotrophic factors and antiinflammatory cytokines have
`
`been shown to play a crucial role in the modulation of
`multiple sclerosis (MS) and its experimental model, experimen-
`tal autoimmune encephalomyelitis (EAE) (1–3). Yet, the infor-
`mation available on their expression within the CNS is limited.
`Various current therapies for MS attempt to interfere with the
`pathological processes by immune intervention and up-
`regulation of these regulatory substances. However, whereas the
`peripheral effect of these treatments has been extensively stud-
`ied, little information exists on their effect in the target organ,
`namely the brain. The study of CNS diseases is particularly
`complicated, because the accessibility of immune factors and
`regulatory cells is strictly controlled by the blood–brain barrier,
`constituting a distinct and unique microenvironment.
`Glatiramer acetate (GA, Copaxone, Copolymer 1), a drug
`approved for the treatment of MS, exerts a marked suppressive
`effect on EAE induced by various encephalitogens in several
`species (4). We previously demonstrated that the therapeutic
`effect of GA is mediated by regulatory cells that suppress disease
`and secrete high amounts of antiinflammatory T helper (Th) 2兾3
`cytokines (5). These GA-induced suppressor cells crossreact
`with the autoantigen myelin basic protein by secreting Th2兾3
`cytokines and induce bystander suppression of processes medi-
`ated by other encephalitogens (6). GA-specific Th2兾3 cells have
`been found in the periphery, in the spleens and lymph nodes of
`experimental animals, and in peripheral blood mononuclear
`cells of humans treated with GA, suggesting that these cells are
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`Immunohistochemical analysis of BDNF expression by GA-specific cells in the brain. Activated labeled GA-specific cells were injected into the peritoneum
`Fig. 1.
`of either EAE-induced (A–C, G–J, and M–O) or normal (D–F) mice. After 7 days, the mice were perfused and brain sections (20 ␮m) were stained immunocyto-
`chemically for BDNF expression. (A–C) Area surrounding the lateral ventricle. (D–F) Choroid plexus in the lateral ventricle. (G–I) Perivascular infiltration in the
`cortex. (J) Overlap image of enlarged Hoechst-labeled, GA-specific cells and their BDNF secretion in the cortex. (M–O) Enlarged images of single GA-specific
`PK-labeled cells in the cortex. Controls demonstrate BDNF expression in corresponding brain regions of an EAE-induced mouse (K) and a normal mouse (L). (Scale
`bar: A–F, K, and L, 100 ␮m; G–I, 20 ␮m; J and M–O, 10 ␮m.)
`
`injections) or had been fed (250 ␮g兾day, eight feedings) with
`GA, respectively, were used throughout this study. Cells were
`selected in vitro by repeating exposures to GA (50 ␮g兾ml) on
`irradiated spleen cells, followed by propagation in T cell growth
`factor medium. These cells had been characterized as Th2
`regulatory cells that can suppress EAE in vivo (5, 6).
`
`T Cell Labeling. GA-specific cells were stained either by Hoechst
`33342-blue fluorescent (Molecular Probes) or by PKH26-red
`fluorescent (Sigma), which incorporate to the cell nucleus or to
`the cell membrane, respectively, according to the manufacturer’s
`instructions. Both staining methods did not interfere essentially
`with the biological activity of the cells, as demonstrated by the
`comparison of the proliferation and cytokine secretion of la-
`beled cells to unlabeled cells and by the ability of the labeled cells
`to prevent EAE.
`
`Adoptive Transfer. Activated prelabeled GA-specific T cells (30 ⫻
`106 per mouse) were injected into the peritoneum of either
`normal or EAE-induced mice 3 days after disease induction by
`whole spinal cord homogenate (5 mg in complete Freund’s
`adjuvant) and pertussis toxin injection (0.2 ␮g per mouse).
`Brains were excised 7 days after cell
`injection because our
`previous studies indicated that this time is optimal for the
`accumulation of GA-specific cells, in contrast to nonspecific
`controls (8, 9).
`
`Immunocytochemistry. Control EAE-induced mice injected with
`GA-specific T cells and normal mice were anesthetized and per-
`fused with 2.5% paraformaldehyde. Brains were sectioned (20 ␮m)
`by cryostat or sliding microtom. Sections were preincubated in PBS
`with 20% horse serum and 0.05% Saponin (Sigma) for 1 h, then
`incubated overnight with the following primary antibodies: chicken
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`Immunohistochemical analysis of IL-10 expression by GA-specific cells in brains of EAE-induced mice. (A–F) Perivascular infiltration in the thalamus. (G–I)
`Fig. 2.
`Enlarged images of single cells in the thalamus. (L–N) Single cells in the cortex. (O–R) Nonoverlapping distribution of IL-10-expressing, GA-labeled cells and
`additional population of cells expressing IL-10 and GFAP in tissue surrounding the lateral ventricle. Controls demonstrate IL-10 expression in corresponding brain
`regions of an EAE-induced mouse (J) and a normal mouse (K). (Scale bar: A–C, 50 ␮m; D–F, 20 ␮m; G–I and L–N, 10 ␮m; J, K, and O–R, 100 ␮m.)
`
`IMMUNOLOGY
`
`anti-BDNF (Promega), goat anti-IL-10 (Santa Cruz Biotechnolo-
`gy), rabbit anti-TGF-␤(Santa Cruz Biotechnology), rat anti-IFN-␥
`(BioSource International, Camarillo, CA), or mouse anti-glial
`fibrillary acidic protein (GFAP; Pharmingen), 1–10 ␮g兾ml, diluted
`in PBS with 2% horse serum and 0.05% saponin. The second
`antibody step was performed by labeling with Cy2- or Cy3-
`conjugated donkey anti-chicken, donkey anti-goat, donkey anti-
`rabbit, donkey anti-rat, or donkey anti-mouse (Jackson ImmunoRe-
`search), 1:250, for 20 min. Additional controls were incubated with
`secondary antibody alone. Slides of Hoechst-labeled cells were
`examined by fluorescence microscope (Nikon Eclipse E600). Slides
`
`of PKH26-labeled cells were examined by confocal microscope
`(Axiovert 100M, Zeiss). Photographs were taken by using SPOT
`software program. Images were processed with PHOTOSHOP (Ado-
`be Systems, Mountain View, CA). Most photographs presented are
`from EAE-induced mice injected with prelabeled GA-specific cells.
`
`Results
`The Presence of Fluorescent-Labeled Adoptively Transferred GA-
`Specific T Cells in the Brain. Abundance of fluorescently prelabeled
`GA-specific cells was observed in brain sections with adoptively
`transferred GA-specific T cells in normal mice and particularly
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`Immunohistochemical analysis of TGF-␤expression by GA-specific cells in brains of EAE-induced mice. (A–D) Perivascular infiltration in the cortex. (G–I)
`Fig. 3.
`3D reconstruction of PK-labeled cells in the thalamus by confocal scanning microscope. (J–M) Nonoverlapping distribution of TGF-␤-expressing GA-labeled cells
`and additional population of cells expressing TGF-␤and GFAP in tissue surrounding a blood vessel. Controls demonstrate TGF-␤in corresponding brain region
`of an EAE-induced mouse (E) and a normal mouse (F) in the cortex. (Scale bar: A–C, 50 ␮m; D, 20 ␮m; E, F, and M–P, 100 ␮m; G–L, 10 ␮m.)
`
`in brains of EAE-induced mice 7 days after their injection to the
`periphery. The GA-specific cells were visible in the ventricles, in
`particular, in the choroid plexus (Fig. 1D) and in the tissue
`surrounding the ventricles (Fig. 1 A). Many clusters of cells were
`seen around blood vessels, indicating perivascular infiltration
`into the brain (Fig. 1G). Individual or clustered cells penetrated
`various regions of the brain, including the cortex, the thalamus,
`the basal ganglia, and the hippocampus. Brain sections, contain-
`ing the penetrating fluorescently labeled GA-specific T cells and
`control brain sections from EAE-induced or normal mice were
`immunohistochemically stained for the expression of the neu-
`rotrophic factor BDNF, the Th2 cytokines IL-10 and TGF-␤, and
`the Th1 cytokine IFN-␥. Images were taken from various regions
`of the thalamus and cortex, mainly from the laterodorsal thal-
`amus nucleus, the ventral posteromedial thalamic nucleus, the
`ventral posterolateral thalamic nucleus, and the somatosensory
`cortex.
`
`BDNF. As shown in Fig. 1, adoptively transferred GA-specific cells
`that had been accumulated in the brain (Fig. 1 A, D, G, and M)
`
`expressed BDNF in situ (Fig. 1 B, E, H, and N). This finding was
`verified by colocalization of the corresponding fields (Fig. 1 C,
`F, I, and O). Furthermore, staining of GA cells in the CNS by
`BDNF antibodies was also demonstrated on the single-cell level
`in Hoechst-labeled (Fig. 1J) and PK-labeled (Fig. 1 M–O) cells.
`GA-specific cells expressing BDNF were observed in various
`regions of the brain, mainly in tissue surrounding the ventricles
`(Fig. 1 A–C) and in perivascular cuffs (Fig. 1 G–I). BDNF
`immunostaining localized both in the cytoplasm and in the
`membrane of the cells (Fig. 1 J and N). In contrast to the strong
`BDNF expression in the brains containing GA-specific cells, only
`marginal BDNF staining was observed within the CNS of control
`EAE-induced (Fig. 1K) and in normal (Fig. 1L) mice into which
`no GA-specific cells were injected.
`
`IL-10. Fig. 2 illustrates that GA-specific cells in the brain (Fig. 2
`A, D, and L) demonstrated intensive staining with anti-IL-10
`antibodies (Fig. 2 B, E, and M), as verified by colocalization of
`the corresponding fields (Fig. 2 C, F, and N). This in situ IL-10
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`IMMUNOLOGY
`
`that had been accumulated in the brain (Fig. 4 A and D) did not
`express IFN-␥ in situ (Fig. 4 B and E). It was not possible to
`detect any IFN-␥in the brains of mice injected with GA-labeled
`cells, neither in EAE-induced (Fig. 4 A–C) nor in normal (Fig.
`4 D–F) mice. On the other hand, intense staining for IFN-␥was
`found in corresponding brain regions of EAE-induced mice that
`were not injected with GA-specific cells (Fig. 4 G and H). This
`staining was much higher than that observed in normal mice
`(Fig. 4I).
`
`Discussion
`Immunomodulating therapies for CNS pathologies have been
`studied extensively and in clinical use for several years. However,
`the understanding of the pathway by which they actually operate
`in the target organ has lagged behind. Thus, neurotrophic factors
`and antiinflammatory cytokines are known to play a crucial role
`in MS and EAE modulation (1–3, 12–15), but information on
`their expression within the CNS is limited. In the present study
`we tested whether treatment with the MS drug GA results in
`secretion of neurotrophic factor and antiinflammatory cytokines
`in situ through specific Th2兾3 cells that penetrate the brain. To
`trace the low amounts of factors secreted by a specific subset of
`T cells on the level of whole-brain tissue, we used the double-
`labeling approach in which prelabeled specific T cells were
`adoptively transferred and their cytokines兾neurotrophic factor
`expression was subsequently immunohistologically detected.
`As clearly demonstrated, GA-specific cells in the brain man-
`ifested intensive expression of the neurotrophic factor BDNF
`(Fig. 1) and of the two antiinflammatory cytokines, IL-10 (Fig.
`2) and TGF-␤(Fig. 3), but no trace of the inflammatory cytokine
`IFN-␥ (Fig. 4). This expression was clearly verified on the
`single-cell level by labeling with two different dyes, i.e., Hoechst
`and PKH26, which incorporate into different cell
`loci (the
`nucleus and the membrane, respectively), indicating that these
`findings do not reflect an artifact caused by cell staining. It has
`been claimed that, because of its ability to bind cellular DNA,
`Hoechst labeling inhibits lymphocyte activity and interacts with
`bystander cells (16). We have not observed these phenomena, as
`demonstrated by the comparison of the proliferation and cyto-
`kine secretion of labeled cells with unlabeled cells and by the
`ability of the labeled cells to prevent EAE (8). Yet, to affirm
`staining specificity, we used, in addition, a vital labeling proce-
`dure by the membrane inserting dye PKH26. Parallel results
`were obtained with both staining methods.
`BDNF, IL-10, and TGF-␤manifested cytoplasmatic as well as
`membranalic expression. Positively stained GA-specific cells
`were apparent in various regions throughout the brain, including
`the cortex, the thalamus, the basal ganglia, and the hippocampus,
`mainly in tissue surrounding the ventricles and in perivascular
`locations. They were found in brains of normal and EAE-
`induced mice after adoptive transfer of GA-specific cells. In both
`cases, staining intensity on a single-cell level looked similar. Yet,
`the total expression on the whole-tissue level was significantly
`higher in EAE-induced mice, because a great deal more GA-
`specific cells were present in the brains of those mice than in the
`brains of normal mice. The difference in cell infiltration could
`result from blood–brain barrier injury caused by the injection of
`pertussis toxin during EAE induction and from the destruction
`typical to the disease process. In any event, an extensive expres-
`sion of these modulatory substances by GA-specific cells in the
`CNS was manifested during the pathological process. T cells
`from lines induced by oral immunization or by daily injections of
`GA demonstrated similar CNS penetration and expression pat-
`terns of all the factors tested.
`Intense staining with BDNF, IL-10, and TGF-␤antibodies was
`found only in brains of mice that had been injected with
`GA-specific cells. In control mice only faint staining was noted.
`It is of interest that BDNF staining in brains of EAE-induced
`
`Lack of expression of IFN-␥by GA-specific cells in the brain. Cells in the
`Fig. 4.
`cortex of an EAE-induced mouse (A–C) and a normal mouse (D–F). Expression
`of IFN-␥in corresponding brain sections of controls: an EAE-induced mouse (G
`and H) and a normal mouse (I). (Scale bar: A–F and H, 40 ␮m; G and I, 100 ␮m.)
`
`expression by GA cells was clearly demonstrated on a single-cell
`level in Hoechst-labeled (Fig. 2 G–I) and PK-labeled (Fig. 2
`L–N) cells. IL-10 was expressed both in the cytoplasm and in the
`membrane of the cells (Fig. 2 H and M).
`Clusters of GA-labeled cells expressing IL-10 were abundant
`in various regions throughout the brain. Moreover, unlabeled
`cells within and in the vicinity of these clusters also expressed
`high levels of IL-10 (Fig. 2 B, C, E, and F). These unlabeled
`IL-10-positive cells had elongated astrocyte-like morphology
`consistent with activated microglia. Triple staining of slides
`containing GA-specific cells (Fig. 2O) for IL-10 (Fig. 2P) and
`GFAP, a component of astrocyte filaments (Fig. 2Q), confirmed
`the astrocyte phenotype of these adjacent cells (Fig. 2R). Thus,
`IL-10 expression was evident not only in the labeled GA-specific
`cells that migrated to the brain but also in the surrounding
`resident CNS cells.
`The intense IL-10 secretion was observed only in brains with
`adoptively transferred GA cells, whereas control mice expressed
`low to moderate IL-10 levels. IL-10 expression in brains of
`EAE-induced mice (Fig. 2 J) was slightly higher than that
`observed in brains of normal mice (Fig. 2K). Moderate IL-10
`expression appeared to be associated with areas of cellular
`infiltrated and perivascular cuffs.
`
`TGF-␤. A similar phenomenon was observed regarding TGF-␤, as
`shown in Fig. 3. Adoptively transferred GA-specific cells in the
`brain manifested extensive TGF-␤ expression (Fig. 3 A–D and
`G–L), with strong cytoplasmatic and membranal staining of
`individual cells (Fig. 3 D and H). The staining pattern was similar
`to the pattern observed for IL-10, including the positive staining
`of surrounding elongated cells of astrocyte morphology, which
`was confirmed by triple labeling with GFAP (Fig. 3 J–M). These
`surrounding bystander resident cells displayed similar or even
`higher TGF-␤expression intensity compared with the staining of
`the GA-specific cell population (Fig. 3D).
`In contrast to the strong TGF-␤ secretion in brains with
`adoptively transferred GA cells, control mice expressed low to
`moderate TGF-␤ levels, which were higher in brains of EAE-
`induced mice (Fig. 3E) than in those of normal mice (Fig. 3F).
`
`IFN-␥. The results presented hitherto pertain to the Th2-related
`cytokines. It was of interest to test the expression pattern of a
`Th1 cytokine such as IFN-␥. As shown in Fig. 4, GA-specific cells
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`mice was marginal and similar to the staining of normal mice
`(Fig. 1 K and L), whereas IL-10 and TGF-␤expression in brains
`of EAE-induced mice were somewhat higher than in brains of
`normal mice (Figs. 2 J and K and 3 E and F). This finding agrees
`with other studies that demonstrated up-regulation of these
`cytokines during disease (1, 2, 14, 15). In contrast to Th2兾3
`cytokine expression, which was increased only marginally in
`brains of EAE-induced mice, a significant augmentation in the
`expression of IFN-␥ was observed in brains of EAE-induced
`mice (Fig. 4 G and H) in comparison with normal mice (Fig. 4I).
`This finding corroborates our previous results, indicating that
`lymphocytes isolated from brains of EAE-induced mice secrete
`high levels of IFN-␥ (8, 9). Notably, this increase in IFN-␥ was
`not manifested in brain lymphocytes from EAE-induced mice
`treated with GA (8, 9) nor in corresponding brain sections from
`EAE-induced mice that had been injected with GA-specific cells
`(Fig. 4B). In both cases, IFN-␥ levels were similar to those of
`normal mice. IFN-␥ is a principal Th1 effector cytokine, which
`by and large is believed to be involved in the pathological
`processes of EAE and MS (1). Hence, the significance of the
`down-regulation of this inflammatory cytokine in the target
`organ, induced by the MS drug GA.
`The current consensus is that activated T cells of any speci-
`ficity cross the blood–brain barrier and penetrate the CNS (18),
`but, thereafter, T cells that are not able to recognize their
`specific antigen in the CNS decline to baseline level. The ability
`of GA-induced T cells to persist in the CNS, while cells with
`different specificity were completely absent, was previously
`shown and attributed to their crossreactivity with the myelin
`antigen myelin basic protein (8, 9). The present study demon-
`strates that GA-specific cells not only accumulate in the brain but
`actually function in the diseased organ as regulatory cells by the
`secretion of Th2兾3 and neurotrophic factor. Of special interest
`is the finding that IL-10 and TGF-␤are expressed not only by the
`GA-labeled cells but also by unlabeled cells within their vicinity
`(Figs. 2 C and F and 3D). These surrounding bystander cells have
`elongated astrocyte-like morphology consistent with activated
`microglia. Staining with GFAP, a component of astrocyte
`filaments, corroborated the astrocyte nature of these cells (Figs.
`
`2 O–R and 3 J–M). The glial cells displayed similar or even higher
`IL-10 and TGF-␤ expression intensity compared with the stain-
`ing of the GA specific population. Such spreading of positive
`staining could result either from genuine IL-10 and TGF-␤
`secretion by the astrocytes that had been activated by the
`cytokines secreted from the GA-specific cells, or from the
`binding of IL-10 and TGF-␤secreted by the GA-specific cells to
`their specific receptors on these astrocytes (17, 19). In both cases,
`this Th2兾3 spreading suggests a bystander therapeutic effect of
`GA on the CNS resident cells.
`IL-10 is a potent regulatory cytokine in autoimmunity that
`inhibits Th1 cells and macrophage activation in addition to its
`effector multifunctional therapeutic reactivity (1, 2, 14). More-
`over, IL-10 can modulate glial cell responses by inhibiting MHC
`class II, NO, and chemokine expression (19). TGF-␤suppresses
`cytotoxic T cell response, production of tumor necrosis factor ␣,
`IFN-␥, and factors that contribute to myelin damage, such as
`lysosomal enzymes and nitrogen intermediates (15). The ability
`of GA-infiltrating cells to express and induce the expression of
`these potent modulating cytokines, also by bystander CNS cells,
`substantiates GA therapeutic activity. Furthermore, the potent
`neurotrophic factor BDNF, expressed by the GA-specific cells in
`situ, is a key regulator of neuronal development, which supports
`neuronal survival and regulates neurotransmitter release and
`dendritic growth (12, 13). BDNF can rescue injured or degen-
`erating neurons and induce axonal outgrowth, remyelination,
`and regeneration (20). The full-length BDNF receptor, tyrosine
`kinase receptor B, has been found in neurons in the vicinity of
`MS plaques and in reactive astrocytes in MS lesions (21). The
`BDNF secreted by the GA-infiltrating cells can therefore actu-
`ally function in the MS兾EAE target tissue. Thus, the expression
`of BDNF, IL-10, and TGF-␤ by GA-specific cells in the CNS
`directly links the therapeutic activity of GA in MS兾EAE and its
`in situ immunomodulatory effect, namely, the antiinflammatory
`bystander suppression and neuroprotection induced by GA-
`specific T cells in the brain.
`
`This study was supported in part by grants from Teva Pharmaceutical
`Industries (Israel) and Terry and Dr. Claude Oster and by a special fund
`of the Eugene Applebaum Family Foundation (Bloomfield Hills, MI).
`
`1. Liblau, R. S., Singer, S. M. & McDevitt, H. O. (1995) Immunol. Today 16,
`34–38.
`2. Kennedy, M. K., Torrance, D. S., Picha, K. S. & Mohler, K. M. (1992)
`J. Immunol. 149, 2496–2505.
`3. Ziemssen, T., Kumpfel, T., Klinkert, W. E., Neuhaus, O. & Hohlfeld, R. (2002)
`Brain 125, 2381–2391.
`4. Teitelbaum, D., Aharoni, R., Fridkis-Hareli, M., Arnon, R. & Sela, M. (1998)
`in The Decade in Autoimmunity, ed. Shoenfeld, Y. (Elsevier, New York), pp.
`183–188.
`5. Aharoni, R., Teitelbaum, D., Sela, M. & Arnon, R. (1997) Proc. Natl. Acad. Sci.
`USA 94, 10821–10826.
`6. Aharoni, R., Teitelbaum, D., Sela, M. & Arnon, R. (1998) J. Neuroimmunol.
`91, 135–146.
`7. Duda, P. W., Schmied, M. C., Cook, S. L., Krieger, J. I. & Hafler, D. A. (2000)
`J. Clin. Invest. 105, 967–976.
`8. Aharoni, R., Teitelbaum, D., Leitner, O., Meshorer, A., Sela, M. & Arnon, R.
`(2000) Proc. Natl. Acad. Sci. USA 97, 11472–11477.
`9. Aharoni, R., Meshorer, A., Sela, M. & Arnon, R. (2002) J. Neuroimmunol. 126,
`58–68.
`
`10. Krakowski, M. L. & Owens, T. (1997) Eur. J. Immunol. 27, 2840–2847.
`11. Kipnis, J., Yoles, E., Porat, Z., Cohen, A., Mor, F., Sela, M., Cohen, I. R. &
`Schwartz, M. (2000) Proc. Natl. Acad. Sci. USA 97, 7446–7451.
`12. Thoenen, H. (1995) Science 270, 593–598.
`13. Barde, Y. A. (1997) Nature 385, 391–393.
`14. Bettelli, E., Nicholson, L. B. & Kuchroo, V. K. (2003) J. Autoimmun. 20,
`265–267.
`15. Morris, M. M., Dyson, H., Baker, D., Harbige, L. S., Fazakerley, J. K. & Amor,
`S. (1997) J. Neuroimmunol. 74, 185–197.
`16. Parish, C. R. (1999) Immunol. Cell Biol. 77, 499–512.
`17. Hulshof, S., Montagne, L., De Groot, C. J. A. & Van Der Valk, P. (2002) Glia
`38, 24–35.
`18. Flugel, A., Berkowicz, T., Ritter, T., Labeur, M., Li, Z., Ellwart, J. W., Willem,
`M., Lassmann, H. & Wekerle, H. (2001) Immunity 14, 547–560.
`19. Ledeboer, A., Breve, J. P., Poole, S., Tilders, F. J. & Van Dam, A. M. (2000)
`Glia 30, 134–142.
`20. Gravel, C., Gotz, R., Lorrain, A. & Sendtner, M. (1997) Nat. Med. 3, 765–770.
`21. Stadelmann, C., Kerschensteiner, M., Misgeld, T., Bruck, W., Hohlfeld, R. &
`Lassmann, R. (2002) Brain 125, 75–85.
`
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`
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