Kinetics and Cellular Origin of Cytokines in the Central Nervous System: Insight into Mechanisms of Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis1 Amy E. Juedes, Peter Hjelmstro¨m, Cheryl M. Bergman, Annie L. Neild, and Nancy H. Ruddle2 Experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 (H-2b) mice is characterized by early (day 12) acute paralysis, followed by a sustained chronic clinical course that gradually stabilizes. Extensive inflammation and demyelination coincide with clinical signs of disease. To identify the mechanisms of these processes, individual proinflammatory and anti-inflammatory cytokines and chemokines were studied. Sensitive single-cell assays were utilized to determine the cellular origin and kinetics of cytokine production in the CNS. Immunization with MOG35–55 peptide resulted in priming of both Th1 (lymphotoxin, IFN-g, and TNF-a) and Th2 (IL-4) cells in the spleen. However, only Th1 cells were apparent in the CNS. CD4 T cells that produced IFN-g or TNF-a were present in the CNS by day 7 after immunization with MOG35–55, peaked at day 20, and then waned. TNF-a was also produced in the CNS by Mac-11 cells. On days 7 and 10 after immunization, the TNF-a-producing Mac11 cells were predominantly microglia. By day 14, a switch occurred in that the Mac11 TNF-aproducing cells had the phenotype of infiltrating macrophages. RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 chemokine mRNA were detected in the CNS by day 8 after immunization. The early presence of monocyte chemotactic protein 1 (MCP-1) in the CNS provides a mechanism for the recruitment of macrophages. These data implicate TNF-a production by a continuum of T cells, microglia, and macrophages at various times during the course of disease. The importance of Th1 cytokines is highlighted, with little evidence for a role of Th2 cytokines. The Journal of Immunology, 2000, 164: 419 – 426.
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he CNS is considered to be an immunologically privileged site with the blood-brain barrier (BBB)3 (3) protecting against infiltrating cells and various high m.w. proteins. In the course of multiple sclerosis and its murine model, experimental autoimmune encephalomyelitis (EAE), the BBB is breached and inflammation ensues. Key issues in neurologic inflammatory diseases are concerned with identifying how this breach occurs and the mechanisms that serve to regulate it. There is considerable evidence that T cells are crucial to these processes. However, the nature of the T cells and their products has not been fully investigated in all models, nor has the interaction of the Agspecific cells, the resident cells, and the additional infiltrating cells been elucidated. Department of Epidemiology and Public Health and Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520 Received for publication August 8, 1999. Accepted for publication October 13, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Multiple Sclerosis Society Grant RG 2394 (to N.H.R.), by a fellowship from the Swedish Foundation for International Cooperation in Research and Higher Education (to P.H.), and by National Institutes of Health Grant TG AI 07019 (to A.E.J.). 2 Address correspondence and reprint requests to Dr. Nancy H. Ruddle, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, P.O. Box 208034, New Haven, CT 06520-8034. E-mail address:
[email protected] 3 Abbreviations used in this paper: BBB, blood-brain barrier; EAE, experimental autoimmune encephalomyelitis; LT, lymphotoxin; MCP, monocyte chemotactic protein; MBP, myelin basic protein; PLP, proteolipid protein; MOG, myelin oligodendrocyte glycoprotein; WT, wild type; IP-10, IFN-inducible protein 10; SSPE, standard saline citrate phosphate/EDTA; ELISPOT, enzyme-linked immunospot; HEL, hen egg lysozyme; SFC, spot-forming cell.
Copyright © 2000 by The American Association of Immunologists
In this communication, we use the myelin oligodendrocyte glycoprotein (MOG) model of EAE to investigate several questions with an eye toward understanding the mechanism by which the integrity of the BBB is broken and the nature of the self-limiting inflammation. C57BL/6 mice are immunized with MOG35–55 peptide in CFA and treated with pertussis toxin and boosted once with the Ag. This immunization regimen results in a reproducible disease course, which in our previous studies demonstrated a 100% incidence of clinical signs (1, 2). Clinical signs of disease are apparent by approximately day 12, peak at ;20 days, and then stabilize, with continued paralysis (1). MOG-induced EAE can be defined as a chronic sustained disease characterized by inflammation and demyelination. Inflammation in this model consists of T cells, B cells, and macrophages (2). In this study, we show that the presence of infiltrating cells precedes clinical signs by a few days, peaks at 20 days, and then becomes less apparent. The MOG model as first described in the rat by Linington and colleagues (3–5) included the presence of Ab specific for the protein. Data obtained with passive transfer experiments implicated both arms of the immune system and were interpreted to indicate that T cells were the initial culprits in breaching the BBB, but that demyelinating Abs were critical for the clinical signs of paralysis. This would suggest the importance of both Th1 (proinflammatory) and Th2 (Ab helper) CD4 T cells. However, this does not appear to be the case in all species since gene-targeted mice that lack B cells or Ab are as susceptible to MOG-induced EAE as their wildtype (WT) littermates (2, 6). In addition, Ab titers to MOG correlate poorly with clinical scores in WT mice (reviewed in Ref. 7), and passive transfer of the disease does occur with T cells alone (see Refs. 8 and 9; A. E. Juedes and N. H. Ruddle, manuscript in preparation). The importance of the Th1 cytokine lymphotoxin 0022-1767/00/$02.00
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(LT)-a (1), but not IFN-g (10), and the lack of importance of the Th2 cytokine IL-4 (9, 11) are suggested by data obtained from gene-targeted mice. However, in a mouse not subject to genetic manipulation, the possibility remained that Th1 and Th2 cytokines do play a role in MOG EAE. Th1 and Th2 cytokine mRNA have been detected in the CNS of mice with EAE induced with recombinant human MOG (12). In this study, we investigate whether murine MOG peptide induces both Th1 and Th2 cytokine proteinproducing cells and whether both of those cell types can penetrate the BBB. The role of nonlymphoid cells such as CNS resident cells and infiltrating macrophages and their products must also be considered in elucidating the mechanisms of EAE. Several studies have addressed the role of macrophages in EAE (13–15), although none used the MOG model. Tran et al. (15) demonstrated the importance of macrophages in an adoptive transfer model of myelin basic protein (MBP)-induced EAE in SJL/J mice. Selective depletion of peripheral macrophages by administration of mannosylated liposomes containing dichloromethylene diphosphonate resulted in greatly reduced clinical severity and reduced invasion of leukocytes into the parenchyma. However, the CNS content of CD4 T cells and microglia was not affected. Martiney et al. (13) used CNI-1493 to inhibit macrophage activation in the same model of EAE, which also resulted in reduced clinical severity and inflammation. In addition, cytokine mRNA levels in the CNS were decreased after macrophage inactivation; however, TNF-a was not examined in that study. TNF-a mRNA production by resident microglia and infiltrating non-T mononuclear CNS cells was demonstrated by Renno et al. (16) in the SJL/J MBP model. The nature of the cells that produce TNF-a has not been addressed in MOG EAE, and in fact evidence conflicts regarding a role for TNF-a in that disease. Liu et al. (17) found that TNFa2/2 mice were even more susceptible to MOG-induced EAE than were WT mice. The data of Suen et al. (1) suggest that LT-a is crucial for EAE and that the presence of TNF-a does not compensate for its absence. However, Korner et al. (18) showed that MOG-immunized TNF-a2/2 mice did have a slightly delayed time of onset. There was also an interesting difference in the pattern of inflammation in the CNS, manifested as a reduction in discrete perivascular cuffs and limited expansion of cells into the parenchyma. The mechanisms by which the LT/TNF cytokines influence inflammation most likely include both induction of adhesion molecules and chemokines. Adhesion molecules such as VCAM-1 and ICAM-1 are up-regulated in several models of EAE, including MOG (6, 19 –22). Murine LT-a and TNF-a are potent inducers of these adhesion molecules in vitro and in vivo (23–25). The expression of chemokines in EAE has been documented by many groups in several models with varying results, although not in the MOG model described here (summarized in Refs. 26 and 27). In many of these studies, analyses have been conducted in the course of inflammation and do not provide a distinction between those chemokines that contribute to inflammation and those that are products of infiltrating cells. LT-a and TNF-a can induce RANTES, monocyte chemotactic protein (MCP)-1, and IP-10 from various cells lines in vitro (24, 28, 29), and LT-a has also been demonstrated to produce those chemokines in vivo in a transgenic model (23). The possibility that such Th1 cytokines influence chemokine expression and later recruitment is examined here. In this communication, EAE is considered as a dynamic process. The cellular origin, the nature, and the temporal pattern of cytokine production in the CNS is investigated and shown in the case of TNF-a to be produced by three different populations, T cells, mi-
croglia, and macrophages. These data provide insight into the mechanisms of this disease.
Materials and Methods Mice Female C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were 8 –10 wk of age at the time of immunization.
MOG peptide MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK), of murine origin, was synthesized by the W. M. Keck Biotechnology Resource Center at Yale University. The peptide was purified by reversed-phase (C18) column HPLC and a trifluoroacetic acid/acetonitrile gradient.
Active induction of EAE EAE was induced by s.c. flank injections of 300 mg MOG35–55 peptide in CFA (Difco, Detroit, MI) with 500 mg Mycobacterium tuberculosis on days 0 and 7, supplemented by i.p. injections of 500 ng pertussis toxin (List Biological Laboratories, Campbell, CA), as described previously (1). Control mice were immunized with 300 mg hen egg lysozyme (HEL) in CFA and injected with pertussis toxin as described above. The mice were observed daily for clinical signs and scored as described previously (1).
Isolation of total RNA/Northern blot analysis Splenocytes were cultured at a concentration of 2 3 106 cells/ml, with or without 20 mg/ml MOG35–55. Cells were harvested 18 h later. Total RNA was isolated using Trizol reagent (Life Technologies, Gaithersburg, MD) according to the instructions provided by the manufacturer. Total RNA (10 mg) was electrophoresed in a 1% agarose/formaldehyde gel with 1 3 MOPS as the running buffer. RNA was transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH), UV cross-linked, and hybridized to random-primed 32P-labeled probes overnight at 42°C as recommended by the manufacturer. After hybridization, the blots were washed twice in 23 standard saline citrate phosphate/EDTA (SSPE), which contained 0.2% SDS, for 30 min at room temperature, and13 SSPE and 0.13 SSPE containing 0.2% SDS for 30 min each at 65°C. The probed blots were exposed at 270°C to Hyperfilm MP (Amersham, Arlington Heights, IL).
cDNA probes A 1.4-kb murine TNF-a cDNA probe (a gift from Dr. Bruce Beutler, University of Texas Southwestern Medical School, Dallas, TX) was excised from the PBS vector with BamHI and PstI. A 0.71-kb KpnI/HincII fragment of the murine LT cDNA was used as the murine LT-a probe as described previously (30).
Bioassay for TNF-a and LT-a TNF-a/LT-a bioactivity was measured by using the WEHI 164 fibrosarcoma cell line as a cytotoxic target as described previously (31). Briefly, serial dilutions of supernatants from splenocytes cultured with or without MOG35–55 were incubated with WEHI 164 cells in 96-well microtiter plates for 48 h at 37°C. Target cell viability was then determined by assessing their metabolic activity with the use of the MTT assay.
Enzyme-linked immunospot analysis The enzyme-linked immunospot (ELISPOT) assay used was that described by Tian et al. (32). To isolate cells from the CNS, mice were deeply anesthetized and perfused intracardially with RPMI 1640 medium (Life Technologies. In each experiment, cells were pooled from two to three equivalently clinically affected mice. Brain and spinal cord cell suspensions were incubated with collagenase II (1 mg/ml; Sigma, St. Louis, MO) at 37°C for 20 min, and mononuclear cells were isolated by discontinuous Percoll (Pharmacia, Piscataway, NJ) gradient. ELISPOT plates (Millipore, Ann Arbor, MI) were coated with the appropriate capture Ab for IFN-g or IL-4. Spleen or CNS cells were plated with or without 10 mg/ml MOG35–55. Plates were incubated at 37°C for 24 h (IFN-g) or 48 h (IL-4) and washed with PBS to remove cells. Biotinylated detection Abs for IFN-g or IL-4 were added and plates were incubated at 4°C overnight. Bound secondary Abs were visualized using HRP-streptavidin (Dako, Carpinteria, CA) and 3-amino-9-ethylcarbazole. Abs R4 – 6A2/XMG1.2-biotin, 11B11/BVD6 – 24G2-biotin (PharMingen, San Diego, CA) were used for capture and detection of IFN-g and IL-4, respectively. Spot-forming cells (SFC) were enumerated with the aid of a dissecting microscope.
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Flow cytometric analysis Cells were isolated from the CNS as described above and cultured at a density of 1–2 3 106 cells/ml at 37°C. Protein transport inhibitors (GolgiStop for IFN-g staining or GolgiPlug for TNF-a staining) were applied as recommended by the manufacturer (PharMingen). Cells were harvested 5 h later and washed in FACS buffer (1% FCS and 0.1% sodium azide in PBS). After blocking with purified rat, hamster, and goat IgG (Pierce, Rockford, IL), cells were stained for surface markers with directly conjugated Abs in FACS buffer. Cells were then fixed, permeabilized, and stained for the intracellular cytokines TNF-a or IFN-g using a Cytofix/ Cytoperm kit (PharMingen) as recommended by the manufacturer. Abs used were CD4-FITC, CD11b (Mac-1)-FITC, CD45-Cy-Chrome, IFN-gPE, and TNF-a-PE. All Abs were obtained from PharMingen.
RNase protection assay Mice were deeply anesthetized and perfused intracardially with PBS. Total RNA was isolated from the brains and spinal cords using Trizol reagent (Life Technologies) according to the manufacturer’s recommendations. Chemokine mRNA levels were determined using the Riboquant Multiprobe RNase Protection Assay System (PharMingen). 32P-labeled riboprobes were synthesized from the plasmid template set mCK-5 using T7 polymerase, after which the DNA template was digested with DNase. Total RNA (15 mg) was hybridized with the riboprobes overnight at 56°C. The following day, ssRNA species were removed by digestion with RNase A. The protected RNA species were phenol/chloroform extracted, ethanol precipitated, and electrophoresed on a 5% polyacrylamide gel. Protected chemokine probes were visualized by autoradiography of the dried gel.
Results Splenocytes produce Th1 and Th2 cytokines in response to immunization with MOG35–55 The phenotype of MOG35–55-specific cells in the spleen was analyzed by evaluating their production of representative Th1 (LT-a, TNF-a, and IFN-g) and Th2 (IL-4) cytokines (33–35). Mice were immunized with MOG35–55 in CFA and pertussis toxin according to the protocol for EAE induction, which as noted above results in clinical signs by day 12. Spleens were removed 14 –20 days after the initial immunization, and cells were cultured with or without MOG35–55. Cytokine protein production was analyzed using a WEHI assay (for TNF-a and LT-a) or an ELISPOT assay (for IFN-g and IL-4). LT-a and TNF-a production were not evaluated by ELISPOT due to the lack of a commercially available Ab that distinguishes between them. Supernatants from cells cultured with MOG35–55 exhibited strong cytotoxic activity against WEHI 164 cells, indicating the Ag-specific production of TNF-a or LT-a protein (Fig. 1A). The production of both cytokines was confirmed at the mRNA level by Northern blot analysis. RNA messages for both TNF-a and LT-a were detected after stimulation with MOG35–55 (Fig. 1B). MOG35–55-specific cells that produced IFN-g, and to a lesser extent IL-4, were also detected by ELISPOT analysis (Fig. 2A). These data indicate that Th1 cells were the predominantly primed T cell population, although Th2 cells were also stimulated. Cells in the CNS and spleen produce a different array of cytokines after MOG stimulation To determine whether both Th1 and Th2 cells infiltrate the CNS, ELISPOT assays were performed for IFN-g and IL-4. Spleen or CNS isolated cells were cultured in medium alone or stimulated with MOG35–55 or anti-CD3. As noted above, both IFN-g and IL-4 protein-producing cells could be detected in the spleen 20 days after immunization in response to MOG35–55 or anti-CD3 stimulation (Fig. 2A). IFN-g-producing cells were also present in the CNS of MOG-immunized mice. They were apparent even in the absence of additional exogenous Ag stimulation and especially at high numbers after stimulation with MOG35–55. Their frequency was 10-fold higher than that seen in the spleen (note difference in scale, Fig. 2C). In contrast, no IL-4-producing cells could be de-
FIGURE 1. Splenocytes produce LT-a and TNF-a in response to MOG35–55. Splenocytes were obtained from two mice (score of 2.5) immunized with MOG35–55 14 days previously. A, LT-a/TNF-a bioassay using supernatants from spleen cells cultured in the presence or absence of MOG35–55. Supernatants from cells cultured with MOG35–55 (F) but not medium alone (E) exhibited cytotoxic activity when cultured with WEHI 164 cells. B, Northern blot analysis of total RNA isolated from splenocytes cultured in the presence or absence of MOG35–55. Blots were hybridized with probes specific for murine TNF-a or LT-a as indicated.
tected in the CNS, even when cells were stimulated in vitro with anti-CD3. At 40 days after immunization, when the clinical signs of disease are stabilized (1), IFN-g-producing cells were still apparent in the CNS, although at a decreased frequency compared with day 20. It was possible that this is due to a switch from a Th1 to a Th2 phenotype. However, this is considered unlikely, because even at this late time, no IL-4-producing cells were detected in the CNS, whereas they were still apparent in the spleen (Fig. 2, B and D). Therefore, there is little support for the concept that Th2 cells contribute to either the induction or resolution of the clinical signs of MOG EAE, as only Th1 cells could be found in the CNS throughout the course of disease. The phenotype of IFN-g- and TNF-a-producing cells in the CNS The frequency and phenotype of cytokine-producing cells in the CNS was evaluated further using intracellular cytokine staining. Using this technique coupled with staining for various cell phenotype markers, it is possible to attribute cytokine production in the CNS to a particular cell type and to evaluate several different cytokines. FACS analysis also allows quantification of the actual number of cytokine-producing cells in the CNS as disease progresses. Cells were isolated from the CNS and cultured for 5 h with or without MOG35–55 and a protein transport inhibitor. Cells were analyzed by FACS after being stained for surface CD4 and intracellular IFN-g, TNF-a, and IL-4 to assess the Th phenotype. The absence of an Ab specific to LT-a prevented the analysis of that cytokine by this technique. Background staining was very low
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FIGURE 2. IL-4 production varies between the CNS and spleen. Cells were pooled from several equivalently affected animals and cultured for ELISPOT analysis in the presence or absence of MOG35–55 or anti-CD3 as indicated. Mice with scores of 3 on day 20 and scores of 2 on day 40 were used. Spleen cells produced SFC for IFN-g (f) and IL-4 (o) after MOG35–55 or anti-CD3 stimulation on day 20 (A) or day 40 (B) after immunization. CNSisolated cells produced SFC for only IFN-g on day 20 (C) and day 40 (D). Representative data from one of three experiments.
for all cytokines in cells isolated from control mice immunized with HEL and pertussis toxin (Fig. 3A). As expected from the ELISPOT data, no IL-4-producing T cells were apparent in mice immunized with MOG. In confirmation and extension of the ELISPOT analyses, MOG35–55-specific IFN-g-producing CD4 T cells were detected in the CNS at several time points after immunization. A representative day 20 experiment is shown in Fig. 3A. MOG-specific T cells were present at a high frequency, representing 17% of total CD4 T cells. IFN-g-producing T cells were also
detected in the CNS after culture with medium alone, although at a much lower level (3%). These cells were presumably activated by presentation of endogenous MOG35–55. MOG35–55-specific T cells in the CNS also produced TNF-a, as shown in Fig. 3A. TNF-a1 MOG-specific T cells were present at a frequency similar to that of IFN-g1 T cells. CD4 T cells were the predominant source of IFN-g in the CNS, although there was a minor population of non-CD4 cells (,1% even at the peak of disease). However, in addition to the T cells that produced TNF-a, we also noticed a substantial population of CD4-negative cells that produced this cytokine. This population of CD4-negative cells expressed Mac-1 (Fig. 3B), a marker for resident CNS microglia and infiltrating macrophages. Mac-11 TNF-a1 cells were detected at a similar frequency (8% of total cells) when cultured with or without MOG35–55. This is in contrast to the Mac-1-negative TNF-a1 population, likely to be CD4 T cells (Fig. 3B). Double staining with Mac-1 and CD4 revealed few cells that coexpressed these molecules (between 0.5 and 1%). Kinetics of TNF-a and IFN-g production in the CNS
FIGURE 3. Mac-11 and CD41 cells produce cytokines in the CNS. CNS cells were pooled from groups of animals after immunization with HEL or MOG35–55 as indicated. Representative data from three independent experiments are shown with the percentage of positive cells in relevant quadrants. A, Cells were isolated 20 days after immunization from three mice, all with disease scores of 3 and cultured in the presence (right column) or absence (left column) of MOG35–55, and stained with CD4FITC and IFN-g-PE or TNF-a-PE as indicated. Plots are gated on CD41 cells. B, CNS cells were isolated 30 days after immunization from three mice, all with disease scores of 3, cultured, and stained with Mac-1-FITC and TNF-a-PE.
The kinetics of cytokine production by different cell types was analyzed throughout the course of disease. By days 7 and 10 after immunization, a few IFN-g-producing CD4 Th1 cells could be detected in the CNS by FACS analysis. These MOG-specific cells represented ;3% of total CD4 T cells and were detected even before clinical signs of disease were apparent (Fig. 4). At these early time points, even though cells were detected by FACS, there was relatively little inflammation apparent in the CNS by hematoxylin and eosin staining. There was a marked increase in the frequency of MOG35–55-specific T cells on days 15 and 20. The peak of MOG35–55-specific T cell infiltration (17% of total CD4 T cells) occurred on day 20 and declined thereafter. By day 40, the number of MOG-specific T cells was significantly reduced to 6%. In fact, at this late time, there was a striking reduction in all of the cell types that infiltrate the CNS. In addition to the FACS data presented here, this was apparent by immunohistochemistry and hematoxylin and eosin staining (data not shown). TNF-a production by T cells was also examined at selected time points and was identical to that of IFN-g. The kinetics of TNF-a1 Mac-11 cell accumulation in the CNS roughly paralleled that of CD4 Th1 cells. Mac-11 TNF-a1 cells were detected at early times (days 7 and 10), peaked at days 15 and 20, and fell off at day 40 (Fig. 4).
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FIGURE 4. Kinetics of cytokine production in the CNS. Cells were isolated from the CNS and analyzed by FACS at various times after immunization with MOG35–55. The percentage of CD4 T cells that were IFN-g1 after culture with MOG35–55 are shown in black bars. The percentage of Mac-11 cells that were TNF-a1 are shown in hatched bars. At each time point, cells were pooled from two to three equivalently affected animals. Average disease scores for each time point are shown in parentheses. Data at each time point are representative of one of several experiments.
Microglia and macrophages produce TNF-a sequentially in the CNS TNF-a1 Mac-11 cells were detected early in the CNS, before the onset of clinical signs of disease. Mac-1 stains both microglial cells as well as blood-derived infiltrating macrophages. We reasoned that microglial-derived cytokines could be important in the early phase of disease in recruiting other cell types into the CNS. To distinguish between parenchymal microglia and blood-derived macrophages, Mac-11 cells were stained with CD45. Parenchymal microglia are Mac-11 CD45low whereas macrophages are Mac-11 CD45high. The CD45high population may also contain some activated microglia, since microglial activation can result in an increase in CD45 expression (36). On days 7 and 10 after immunization, .60% of the Mac-11 cells were CD45low microglia, and they were the primary source of Mac-11-derived TNF-a (Fig. 5). The TNF-a production by Mac-11 cells in the CNS was dependent upon immunization with MOG35–55, since few cells isolated from mice immunized with HEL and supplemented with pertussis toxin were TNF-a1 (Fig. 5A). Around day 14 or 15 after MOG immunization, there was a switch in the proportion of CD45high Mac-11 cells, which increased to ; 80% (Fig. 5A). These cells produced TNF-a (Fig. 5B). The influx of macrophages into the CNS closely paralleled the pattern of T cell infiltration, with day 15 also marking a large increase in the number of MOG35–55-specific T cells in the CNS. Macrophage infiltration continued, and, by day 20, most of the Mac-1-derived TNF-a was produced by CD45high macrophages. By day 40, the frequency of Mac-11 CD45high cells was reduced, and this resulted in a corresponding decrease in the percentage of TNF-a that was derived from this population (Fig. 5B). Note that late in the disease, there was a low level of TNF-a present in the CNS (Fig. 4), and this reduced amount was produced by resident microglia (Fig. 5B). A restricted set of inflammatory chemokines is expressed early in the CNS Experiments were conducted to investigate the mechanism by which the large influx of macrophages into the CNS occurred. We
FIGURE 5. Microglia and macrophages produce TNF-a in the CNS at different times during the course of MOG-induced EAE. Cells were isolated from the CNS of equivalently affected animals (from Fig. 4) at various days after immunization with HEL or MOG35–55 as indicated and stained with Mac-1-FITC, CD45-Cy-Chrome, and TNF-a-PE. Data are gated on Mac-11 cells. A, Representative plots from days 7, 10, 14, and 15. B, Graph of the percentage of total Mac-11 TNF-a that is derived from macrophages (E) or microglia (f) at various days after immunization. All data are representative of one of several experiments.
investigated the possibility that there was an up-regulation of a macrophage-specific chemokine in the CNS at an early time after immunization. RNase protection assays on total RNA isolated from the brain and spinal cord performed day 8 after immunization with MOG35–55 showed expression of MCP-1, RANTES, and IP-10 (Fig. 6). These chemokines were apparent even before extensive mononuclear cell infiltration and could be of parenchymal origin. The increase in CNS chemokine expression was dependent upon immunization with MOG, since neither normal animals (Fig. 6) nor those immunized with CFA and treated with pertussis toxin (data not shown) expressed any of the chemokines detected with this probe set. With overexposure of the autoradiogram, MIP-1a was also weakly detected in one experiment 10 days after immunization with MOG35–55. At later time points, the pattern of chemokine expression was considerably more complex in that mRNAs for many additional chemokines, including MIP-1a, MIP-1b, and MIP-2, could be detected (data not shown). The array was quite similar to that seen when RNA was analyzed from total lymph node cells, suggesting that later in disease, the infiltrating cells themselves were contributing to chemokine expression.
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FIGURE 6. MCP-1, RANTES, and IP-10 are expressed in the CNS 8 days after immunization with MOG35–55, before the onset of clinical signs. RiboQuant multiprobe RNase protection system with template set mCK-5 was used to determine the expression of chemokines after MOG immunization. The samples were probe, yeast tRNA, or RNA extracted from the CNS of healthy controls or mice immunized with MOG35–55 as indicated.
Discussion MOG-induced EAE is a nonrelapsing, chronic sustained disease. In this paper, we investigate the initiation and regulation of inflammation by considering how the privilege of the CNS is broken. We show that immunization with MOG results in priming of both Th1 and Th2 cells in the spleen. With an array of single-cell assays, we demonstrate that the nature of the Ag-specific cells that accumulate in the CNS differs from that in the periphery, with exclusive detection of Th1 cytokines throughout disease, even as the clinical signs and inflammation wane. The data provide evidence for TNF-a production from different cell types at distinct times in the disease process. Early on, CD4 T cells and microglia predominate, whereas later there is continued expression by CD4 T cells and a switch to macrophages. At the peak of disease, CD45high macrophages outnumber microglia. This has also been demonstrated to occur in rat MBP-induced EAE using bone marrow chimeras, where hematogenous donor macrophages outnumbered resident host-derived microglia in the spinal cord 4- to 7-fold (37). The early expression of the chemokines MCP-1, RANTES, and IP-10 in the CNS provides a mechanism for the later influx of macrophages and T cells. All three of these chemokines attract T cells (38 – 40). In addition, MCP-1, and to a lesser extent RANTES and IP-10, also attract monocytes (41– 43). The analysis of cytokines in the CNS reveals production of IFN-g and TNF-a by CD4 Th1 cells that parallels the disease course with a peak around day
20 and then a gradual waning. These data do not reveal substantial production of these cytokines by CD8 cells, NK cells, or B cells. Cytokine production by astrocytes was not examined here, since the protocol for isolating CNS mononuclear cells excludes them (44). The kinetics of TNF-a production is particularly interesting in that this cytokine was produced not only by infiltrating T cells, but also early on by microglia. There was a later switch to TNF-a production by macrophages and then the decline seen in T cells. The population defined here as CD45high macrophages may also contain some activated microglia, since microglial activation can result in an increase in CD45 expression (36). However, we consider this to be unlikely since the same study demonstrated that the extent of the increase in CD45 expression is not sufficient to result in an overlap of the two populations. Several previous studies have addressed cytokine and chemokine production in EAE, although most have considered them in non-MOG models, and none have examined them in detail at the kinetic and cellular level employed here. Our results indicating a poor representation of Th2 cytokines in the spleen, and the absence in the CNS, once again suggest that MOG EAE in the mouse is not dependent on Ab production (2, 6). Several studies have suggested that IL-4 plays a down-regulatory role in EAE (45– 47), although none were conducted with the MOG model. Our studies with that model do not support a role for IL-4 and are in agreement with that of Di Rosa et al. (48), who showed that IL-4 production by CNSinfiltrating cells was low or undetectable during remission of EAE induced with PLP139 –157 in SJL mice. We have not found convincing evidence that certain other cytokines that have been implicated either positively or negatively in EAE play a role in mice not subject to genetic manipulation. These additional cytokines include IL-6 and IL-10. IL-62/2 mice are completely resistant to clinical signs of the disease (6, 49 –51), whereas IL-102/2 mice are even more susceptible, developing a more severe EAE than their WT littermates. Using the FACS analysis employed here, we have not detected any significant expression of IL-6 or IL-10 protein in the CNS. Other studies have evaluated cytokine mRNA production in the CNS during EAE. Renno et al. (16) evaluated cytokine mRNA by RT-PCR from cells isolated from the CNS 17 days after immunization of SJL mice with MBP. They report that T cells in the CNS produce IFN-g and IL-2, but not TNF-a. This is in contrast to our studies that demonstrate TNF-a protein production by CD4 T cells. In agreement with our studies, Renno et al. (16) demonstrate TNF-a mRNA in CNS microglia and macrophages. However, they did not detect the switch in populations noted in our studies, in all probability because they concentrated on only one time point. Okuda et al. (12) examined cytokine mRNA expression in the CNS of mice with EAE induced by immunization with recombinant human MOG. Similar to our studies, they detected the presence of mRNA for the Th1 cytokines TNF-a, LT-a, and IFN-g. However, contrary to our studies, they reported the presence of the Th2 cytokines IL-4 and IL-10 at the severe early phase of the disease and also noted a further increase in IL-10 in the chronic late phase of disease. This discrepancy could be due to the different Ags employed, or to the different techniques used to examine cytokine expression. Okuda et al. (12) used PCR-based methods, which are extremely sensitive, and examined cytokine mRNA and not protein. Additionally, they were using recombinant human MOG protein and not murine peptide to induce disease. As noted above, several studies have evaluated chemokines in EAE, but none thus far in the model of MOG EAE described here. The general conclusion of these previous studies is that several chemokines are activated in a pattern that varies temporally and spatially. In several studies, MIP-1a is detected after disease onset
The Journal of Immunology and shown to correlate with EAE severity, with less emphasis placed on MCP-1, RANTES, and IP-10 (52, 53). In addition, disease induced by adoptive transfer of PLP T cells can be prevented by treatment with an Ab to MIP-1a, suggesting a crucial role for this chemokine (52). In other studies, the chemokines MCP-1, RANTES, and IP-10 are activated. In the report by Berman et al. (54), MCP-1 protein was detected at the onset of inflammation in myelin-induced EAE in the Lewis rat . The MCP-1 expression was interpreted to correlate with the late influx of macrophages. These authors conclude that MCP-1 is a mediator of macrophage infiltration in EAE, a conclusion supported by our data. Two other studies implicate MCP-1 in multiple sclerosis (55, 56). An additional study using a variant of the MOG model implicates C10 in macrophage recruitment (27). This chemokine was not analyzed here. The cellular origin of chemokines in all models and the nature of several additional lymphoid organ specific chemokines has not been determined. The data provided here, taken along with those obtained with gene-targeted mice, provide a framework for elucidating the process of inflammation in EAE. We define three nearly simultaneous early events: Th1 T cell invasion, chemokine activation, and TNF-a production by microglia. Which event is first? Are the processes independent? We propose that the early infiltrating Th1 cells are key to the later events. Frei et al. (57) have shown that IFN-g, one of the Th1 products analyzed here, activates microglia to produce TNF-a, and Renno et al. (16) have demonstrated that Th1 cytokine supernatants induce TNF-a mRNA from CD45low CNS cells. We suggest that MOG-activated T cells enter the CNS and activate microglia which produce TNF-a, leading to the induction of VCAM and ICAM. These adhesion molecules facilitate the traffic of mononuclear cells across the endothelium. This may explain the phenomenon seen in MOG-immunized TNF-a2/2 mice. It may be that the initial production of TNF-a is crucial for the movement of cells into the parenchyma. In its absence, cells remain in the perivascular region. Nearly concurrent with T cell invasion and TNF-a production by microglia is the induction of chemokines. As noted above, LT-a and TNF-a induce MCP-1 in vitro, and these cytokines are likely to induce MCP-1 to attract the later influx of macrophages. Macrophages also produce TNF-a, inducing more adhesion molecules and infiltration. One question not answered here is how the process of inflammation in EAE is limited. Despite the continued presence of Th1 Ag-specific cells in the spleen at 40 days, these cells are no longer found at high levels in the CNS. There are several nonmutually exclusive explanations for this limitation. Although IL-4 was not induced late in disease, it is possible that other anti-inflammatory cytokines such as TGF-b may be induced. This could occur in combination with a lack of recruitment into the CNS late in disease due to a decrease in the expression of chemokines and adhesion molecules. Another possibility is the exhaustion of infiltrating cells due to apoptosis. Many studies have demonstrated apoptosis of infiltrating cells in the CNS of animals with EAE (58 – 60). Recently, a study was published that demonstrated a role for the Fas ligand in the recovery from EAE. Sabelko-Downes et al. (61) showed that Fas ligand-deficient recipients of WT MBP-specific lymphocytes developed prolonged clinical signs of disease. In addition, these mice had an increased number of CD4 T cells present in the CNS late in disease, suggesting a role for Fas ligand in curtailing the expansion of activated Fas1 lymphocytes. It has also been postulated that B cells or their products could play a protective immunoregulatory role in EAE (reviewed in Ref. 7). The limitation of inflammation seen in the CNS could also be the result of a decrease in Ag-presenting capability due to lower levels of MHC
425 II or costimulatory molecules. The capabilities of APC in the CNS at various times in disease are currently under investigation. The data provided here emphasize that EAE is a dynamic process with ordered sequential contributions by individual cells and their products. One of the more fascinating observations is that TNF-a is produced by three different populations of cells at different times in the disease. The data provide several potential cellular, chemokine, and cytokine targets for therapeutics that affect the disease at different times in the process, allowing the lessons learned from this model to be applied to human inflammatory diseases.
Acknowledgments We thank Drs. Paul Lehmann and Oleg Targoni for assistance with the ELISPOT assay.
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