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The Journal of Immunology, 2000, 164: 2759-2768.
Copyright © 2000 by The American Association of Immunologists

IFN-{gamma} Shapes Immune Invasion of the Central Nervous System Via Regulation of Chemokines1

Elise H. Tran, Esther N. Prince and Trevor Owens2

Neuroimmunology Unit, Montreal Neurological Institute, and Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dynamic interplay between cytokines and chemokines directs trafficking of leukocyte subpopulations to tissues in autoimmune inflammation. We have examined the role of IFN-{gamma} in directing chemokine production and leukocyte infiltration to the CNS in experimental autoimmune encephalomyelitis (EAE). BALB/c and C57BL/6 mice are resistant to induction of EAE by immunization with myelin basic protein. However, IFN-{gamma}-deficient (BALB/c) and IFN-{gamma}R-deficient (C57BL/6) mice developed rapidly progressing lethal disease. Widespread demyelination and disseminated leukocytic infiltration of spinal cord were seen, unlike the focal perivascular infiltrates in SJL/J mice. Gr-1+ neutrophils predominated in CNS, and CD4+ T cells with an activated (CD69+, CD25+) phenotype and eosinophils were also present. RANTES and macrophage chemoattractant protein-1, normally up-regulated in EAE, were undetectable in IFN-{gamma}- and IFN-{gamma}R-deficient mice. Macrophage inflammatory protein-2 and T cell activation gene-3, both neutrophil-attracting chemokines, were strongly up-regulated. There was no induction of the Th2 cytokines, IL-4, IL-10, or IL-13. RNase protection assays and RT-PCR showed the prevalence of IL-2, IL-3, and IL-15, but no increase in IL-12p40 mRNA levels in IFN-{gamma}- or IFN-{gamma}R-deficient mice with EAE. Lymph node cells from IFN-{gamma}-deficient mice proliferated in response to myelin basic protein, whereas BALB/c lymph node cells did not. These findings show a regulatory role for IFN-{gamma} in EAE, acting on T cell proliferation and directing chemokine production, with profound implications for the onset and progression of disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Entry of immune cells into and their retention and activation within tissues are crucial features of host immune response against pathogens and of autoimmune pathogenesis. A complex interplay among cytokines, chemokines, and adhesion molecules orchestrates these cellular events and shapes the outcome of an inflammatory reaction. In general, the CXC or {alpha}-chemokines, including IL-8, KC, and macrophage inflammatory protein-2 (MIP-2),3 act primarily on neutrophils, while the CC or ß-chemokines (e.g., MCP-1, MIP-1{alpha}, MIP-1ß, and RANTES) act mainly on monocytes/macrophages and lymphocytes (1, 2). IFN-{gamma}, the prototypic Th1 cytokine, plays an important role in protective, cell-mediated immunity (1, 3). In many organ-specific autoimmune diseases, however, IFN-{gamma} is implicated in pathology (1, 2, 3). Th1 cytokine-producing CD4+ T cells induce organ-specific autoimmune responses characterized by mononuclear infiltrates in the target tissue. Recently, several chemokines have been shown to have selective effects on subsets of CD4+ T cells. Th1 CD4+ T cells respond preferentially to RANTES, MIP-1{alpha}, and MIP-1ß, while Th2 cells respond to TCA-3 (4, 5, 6). By contrast, Th2 cytokines, such as IL-4, IL-5, IL-10, and IL-13, are implicated in pathologic allergic responses that are dominated by eosinophilia or neutrophilia (1, 7). These cytokines have been implicated in amelioration of autoimmune disease or remission (1, 8, 9, 10, 11). In immunocompromised hosts, Th2 CD4+ T cells can induce autoimmunity (12, 13). Infiltrates in the affected tissues of Th2-induced recombinase-activating gene-deficient mice with autoimmune disease included many granulocytes (12, 13).

In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), Th1 CD4+ T cells infiltrate the CNS (14). Chemokines, including RANTES, MIP-1{alpha}, MCP-1, and IFN{gamma}-inducible protein-10 (IP-10), and chemokine receptors are up-regulated in MS and EAE, and their expression has been shown to correlate with the distribution of CNS inflammatory infiltrates and clinical disease activity (15, 16, 17, 18, 19, 20, 21, 22). Anti-MIP-1{alpha} mAb treatment and vaccination with naked DNA encoding MIP-1{alpha} and MCP-1 prevented EAE in rodents (23, 24), and anti-MCP-1 mAb treatment attenuated relapses of EAE (25). EAE can be induced by various myelin components, such as myelin basic protein (MBP), and by adoptive transfer of T cells reactive to these components. SJL/J (H-2s) is a widely used susceptible strain to MBP-induced EAE, while strains such as BALB/c (H-2d) and C57BL/6 (H-2b) are resistant to induction of EAE by immunization with MBP/CFA (14). However, IFN-{gamma}-deficient mice on resistant BALB/c and C57BL/6 background are susceptible to MBP-induced EAE (26, 27). Similarly, Willenborg et al. (28) reported that MOG35–55 induced EAE with high mortality in otherwise resistant 129sv mice lacking IFN-{gamma}R. Neutrophilia was noted in the CNS of IFN-{gamma}R-/- mice with MOG-induced EAE (28).

Dynamic interplay between cytokines and chemokines may direct the trafficking and recruitment of selective leukocyte subpopulations to the tissue sites of inflammation. In this study we explored the cellular and molecular mechanisms underlying CNS autoimmune disease in the absence of the prototypic Th1 cytokine IFN-{gamma} or its receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free female SJL/J and BALB/c mice (8–10 wk old) were purchased from Charles River Canada (St. Constant, Canada). Heterozygous BALB/c-backcrossed mice with the disrupted IFN-{gamma} gene were obtained from Genentech (South San Francisco, CA) (29). These heterozygotes were intercrossed in our animal facility, and the progeny were genotyped by PCR amplification of tail DNA according to the method of Goes et al. (30). Only homozygotes (IFN-{gamma}-/-) were used in this study. C57BL/6-backcrossed mice with the disrupted IFN-{gamma}R were obtained from Dr. Michel Aguet (Institute of Molecular Biology I, Zurich, Switzerland) (31). They were further crossed to the C57BL/6 background in our animal facility and screened by PCR of genomic DNA as previously described (31). The heterozygotes were interbred to yield homozygous (IFN-{gamma}R-/-) mice. All mice with disrupted IFN-{gamma}R used in this study were homozygous (IFN-{gamma}R-/-), and wild-type littermates (IFN-{gamma}R+/+) were used as controls.

Induction of EAE

EAE was elicited by s.c. immunization (base of tail) with an emulsion containing 400 µg of bovine MBP (prepared as described by Cheifetz et al. (32) or purchased from Sigma (Montreal, Canada)) and 50 µg of Mycobacterium tuberculosis H37RA (Difco, Detroit, MI) in CFA (Difco) and boosted in the flanks 7 days later with the same amount. Mice were monitored daily for clinical signs of EAE that was scored as: 1) flaccid tail, 2) hindlimb weakness and poor righting ability, 3) inability to right and one hindlimb paralyzed, 4) both hindlimbs paralyzed with or without forelimb paralysis and incontinence, and 5) moribund. All mice were kept in specific pathogen-free environment. Animal breeding and maintenance and all experimental protocols were in accordance with the Canadian Council for Animal Care guidelines and were approved by McGill University animal care committee.

In vitro proliferation assay of lymph node cells (LNC)

A single cell suspension was prepared from the draining lymph nodes 14 days after the first immunization, and cells (4 x 106/ml) were cultured for 4 days in 200 µl/well with or without 50 µg/ml MBP in RPMI 1640 (Life Technologies, Burlington, Canada) supplemented with 10% FCS (Upstate Biotechnology, Lake Placid, NY), 50 mM 2-ME (Sigma), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin (Life Technologies), and 100 µg/ml streptomycin (Life Technologies). Cultures were pulsed with 0.5 µCi of [3H]thymidine/well (ICN Biochemicals, Mississauga, Canada) during the last 18 h of incubation. [3H]thymidine uptake was measured as counts per minute.

Histology and immunohistochemistry

Mice were anesthetized with sodium pentobarbital (MT Pharmaceutical, Cambridge, Canada) and perfused intracardially through the left ventricle with ice-cold PBS for OCT-embedded tissues or followed by 10% buffered formalin for paraffin-embedded tissues. One-micron paraffin sections were stained with hematoxylin and eosin (H&E) or Luxol Fast Blue to assess demyelination. Immunohistochemical staining was performed on 10-µm cryostat sections. Frozen sections were blocked in 5% normal rabbit serum (Vector, Mississauga, Canada) in PBS for 30 min at room temperature and incubated with primary rat mAbs for 1 h at room temperature or overnight at 4°C, then with biotinylated rabbit anti-rat Ig (Vector) for 1 h at room temperature. Sections were treated with 0.3% H2O2 to quench endogenous peroxidase activity, then incubated with an avidin-HRP complex (Vectastain ABC kit, Vector) following the manufacturer’s instructions. Biotin-avidin complex binding was detected by the use of diaminobenzidene (Medicorp, Montreal, Canada) as chromagen. The mAbs used were GK1.5 (CD4; American Type Culture Collection, Manassas, VA), F4/80 (provided by Dr. Georg Kraal, Vrije Universiteit, Amsterdam), M1/70 (Mac-1/CD11b; American Type Culture Collection), P7/7.1 (MHC II, American Type Culture Collection), RB6-8C5 (Gr-1/Ly6G; PharMingen, San Diego, CA), and MEC 13.3 (CD31/PECAM-1; PharMingen). Control sections were incubated with isotype-matched primary Abs or with secondary Abs alone. Staining for iNOS using polyclonal anti-mouse iNOS (Transduction Laboratories, Lexington, KY) was performed as previously described (33).

Electron microscopy

Mice were perfused with PBS, followed by 0.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Tissues were postfixed in 2% osmium tetroxide, dehydrated in graded concentrations of methanol, cleared in propylene oxide, and embedded in Epon. Ultrathin sections of spinal cord were mounted on nickel grids, stained with uranyl acetate and lead citrate, and examined by electron microscope.

Flow cytometric analysis

After perfusion with ice-cold PBS, brains were removed, and spinal cords were dissected from the vertebral canal, taking care to collect the meninges. Isolation of cells from the CNS was performed as previously described (34). Briefly, tissues were dissociated in RPMI 1640/10% FCS by passing through a metal sieve, then centrifuged at 400 x g for 10 min at 4°C. The pellet was resuspended in 70% isotonic Percoll (Pharmacia, Piscataway, NJ), overlaid with equal volumes of 37 and 30% isotonic Percoll, and centrifuged at 500 x g for 20 min at room temperature. Cells were collected from the 37:70% interface and washed with RPMI 1640/10% FCS. Cells were first incubated on ice for 30 min with 100 µg/ml normal rat Ig in 2.4G2 (anti-Fc{gamma}RIIb/III) supernatant to block Fc receptors and avoid nonspecific staining, then double stained with PE-conjugated anti-CD4 (PE-CD4; Becton Dickinson, Mississauga, Canada) and fluorescein-conjugated anti-CD3 (FITC-145.2C11), FITC-conjugated anti-CD69 (PharMingen), or biotinylated anti-CD25 (PC61, American Type Culture Collection) mAbs, which were visualized by FITC-coupled streptavidin. Cells were also double stained with fluorescein-conjugated anti-Mac-1/CD11b (M1/70) and biotinylated anti-B7.2/CD86 (GL1, PharMingen), which were visualized by PE-coupled streptavidin. Cells were analyzed using a FACScan (Becton Dickinson). Propidium iodide staining and forward/side scatter gating were used to exclude dead cells.

RNase protection assay (RPA)

Total RNA was purified from homogenized PBS-perfused CNS using Trizol (Life Technologies) following the manufacturer’s instructions. Multiprobe DNA templates for chemokines (lymphotactin, RANTES, eotaxin, MIP-1{alpha}, MIP-1ß, MIP-2, IP-10, MCP-1, and TCA-3), cytokines (IFN-{gamma}, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-13, and IL-15), and the housekeeping genes, L32 and GAPDH, were all purchased from PharMingen. RPA was performed according to the manufacturer’s protocol. Briefly, the DNA templates were used to synthesize antisense riboprobes, which were labeled with [{alpha}-32P]UTP (DuPont-NEN Research Products, Guelph, Canada) using T7 polymerase. Labeled probes were hybridized with 20 µg of total RNA at 56°C for 16 h. Samples were then digested with RNase A and T1, and treated with proteinase K. The remaining RNase-protected RNA duplexes were extracted with phenol/chloroform/isoamyl alcohol (Life Technologies) and resolved on 5% denaturing polyacrylamide gels. Undigested labeled probes were loaded in the gels to serve as size markers. Dried gels were visualized by autoradiography or PhosphorImager (Molecular Dynamics, Sunnyvale, CA) after an exposure of 12–48 h for chemokines and 4–7 days for cytokines.

RT-PCR

RNA was reverse transcribed with 10 µM random hexamer primers (Roche), 0.5 mM each of dNTPs (Pharmacia), 3.3 mM DTT (Life Technologies), and 400 U of Moloney murine leukemia virus RT (Life Technologies) at 42°C for 1 h; this was terminated by heating at 75°C for 10 min.

PCR conditions were optimized for linear amplification to allow direct comparison between samples. Equal amounts of cDNA were amplified using 1x PCR buffer (Sigma), 2 mM MgCl2 (Sigma), 80 µM each of dNTPs, 2 U of Taq polymerase (Sigma), and 50 pmol of each primer. The PCR reaction was performed with a Perkin-Elmer (Mississanga, Canada) thermocycler for 30 cycles with denaturation/annealing/extension conditions optimal to each primer set: IL-10 (94°C for 45 s, 58°C for 45 s, and 72°C for 1 min), IFN-{gamma} (94°C for 30 s, 65°C for 30 s, and 72°C for 1 min), IL-12p40 (94°C for 45 s, 63°C for 45 s, and 72°C for 1 min), and ß-actin. The primer sequences for IL-10 were: sense, 5'-TGGCTCAGCACTGCTATGCT-3'; and antisense, 5'-ATGGCCTTGTAGACACCTTG-3'. The other primer sequences were described previously as follows: IFN-{gamma} and ß-actin (35), and IL-12p40 and IL-4 (36). For IL-4 and ß-actin duplex RT-PCR, cDNA were amplified with 1x PCR buffer (Sigma), 4 mM MgCl2 (Sigma), 120 µM each of dNTPs, 4 U of Taq polymerase (Sigma), 50 pmol of each IL-4 primer, and 10 pmol of each ß-actin for 30 cycles (94°C for 45 s, 56°C for 45 s, and 72°C for 1 min). PCR products were electrophoresed in a 1.8% agarose gel, visualized by ethidium bromide or SYBR Green staining (Molecular Probes, Eugene, OR), and analyzed by PhosphorImager (Molecular Dynamics).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma}-/- and IFN-{gamma}R-/- mice develop lethal EAE following immunization with MBP

Immunization with MBP induces an MS-like relapsing-remitting course of disease in SJL/J (H-2s) mice. BALB/c (H-2d) and C57BL/6 (H-2b) mice are resistant to MBP-induced EAE (14, 37). However, whether on a susceptible or a resistant background, mice lacking IFN-{gamma} have been shown to develop EAE with unusually high mortality following immunization with MBP (26, 27, 38). In Table IGo, we show that MBP also induced lethal EAE in mice deficient in IFN-{gamma}R on a C57BL/6 background, while their wild-type littermates were resistant. All IFN-{gamma}-/- and IFN-{gamma}R-/- mice exhibited complete paralysis of both fore- and hindlimbs and immobility, and they died or were euthanized within 24–48 h after onset. By contrast, SJL/J mice showed a wide range of severity, ranging from mild to severe disease, but all of them remitted. As we have consistently observed (39, 40), some of the SJL/J mice that remitted showed a relapsing progression. IFN-{gamma}R-/- mice immunized with CFA alone did not develop signs of neurological deficit (Table IGo). The rapidly progressive course of disease and lethality observed in both IFN-{gamma}-/- and IFN-{gamma}R-/- mice are not features of typical relapsing-remitting EAE, suggesting unique CNS pathology related to the lack of IFN-{gamma} response.


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  		Table I. MBP-induced EAE in IFN{gamma}-/-, IFN{gamma}R-/- mice, wild-type littermates, and SJL/J mice

 
Disseminated neutrophil invasion and demyelination in IFN-{gamma}-/- and IFN-{gamma}R-/- mice

To understand how IFN-{gamma} influences histopathology, we compared mice that lack IFN-{gamma} response (IFN-{gamma}-/- and IFN-{gamma}R-/- mice) to SJL/J mice in which IFN-{gamma} was up-regulated during EAE and its receptor was intact. Neither BALB/c nor C57BL/6 mice developed EAE following MBP/CFA immunization; therefore, they could not be used in our comparative study of CNS pathology. To characterize the acute lethal disease provoked in IFN-{gamma}-/- or IFN-{gamma}R-/- mice, CNS inflammation and demyelination were assessed. Findings in IFN-{gamma}-/- and IFN-{gamma}R-/- mice were indistinguishable from each other with respect to all parameters examined. H&E staining of spinal cord and brainstem from IFN-{gamma}-/- mice with fulminant clinical signs of EAE revealed extensive infiltration by cells morphologically identifiable as polymorphonuclear leukocytes (PMN; Fig. 1GoA). The cerebellum was not affected. This contrasts with acute nonlethal EAE, whether mild or severe, in SJL/J mice. Infiltration in SJL/J mice was characterized by perivascular cuffs containing predominantly mononuclear cells (Fig. 1GoB) and occurring in spinal cord, brainstem, and cerebellum.



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  		FIGURE 1. Spinal cord histopathology of IFN-{gamma}-/-, IFN-{gamma}R-/-, and SJL/J mice with EAE on days 16–17 postimmunization. A and B, H&E staining of paraffin sections from an IFN-{gamma}-/- mouse (A) and an SJL/J mouse (B) with EAE. Note the disseminated infiltration of PMN (arrows) into the parenchyma away from the inflamed vessel in IFN-{gamma}-/- mice (A), and perivascular cuffs of mononuclear cells in SJL/J mice (B). C and G, Immunohistochemical staining using anti-Mac-1/CD11b mAb on frozen sections of an IFN-{gamma}-/- mouse (C) and an SJL/J mouse (G). D, E, F, and J, Anti-Gr-1 staining on frozen sections of IFN-{gamma}-/- (D), SJL/J (E), and IFN-{gamma}R-/- (F) mice and higher magnification of an infiltrate stained for Gr-1 in an SJL/J mouse (J). Note that infiltrates in SJL/J mice contained many Mac-1+ cells but fewer Gr-1+ cells (G vs J). H and K, Anti-F4/80 staining in an SJL/J (H) and an IFN-{gamma}-/- (K) mouse. I and L, Anti-CD31/PECAM-1 in an SJL/J mouse (I) and an IFN-{gamma}-/- mouse (L). All immunohistochemical stainings were revealed using DAB as chromagen and were counterstained with Harris’ hematoxylin, except in I and L, in which aminoethylcarbazole was used as chromagen and counterstained with Mayer’s hematoxylin. Original magnification: A, B, H, and K, x180; C–F, x20; G, I, J, and L, x200.

 
The presence of PMNs was further confirmed by immunohistochemical staining using the RB6-8C5 Ab that recognizes Gr-1/Ly6G on neutrophils (41). Fig. 1Go, D and F, shows widespread, disseminated infiltration by Gr-1+ neutrophils that formed confluent hypercellular areas in the meninges, white matter, and extending into gray matter of IFN-{gamma}-/- and IFN-{gamma}R-/- mice with fulminant EAE. Although Gr-1+ cells could be identified within the vascular lumen and perivascularly, they did not appear to be restrained within defined cuffs around PECAM-1+-activated endothelial cells (Fig. 1GoL). Staining for Mac-1 was also widely disseminated in IFN-{gamma}-/- spinal cord (Fig. 1GoC). In SJL/J mice with paralytic EAE on day 16 postimmunization, neutrophils were also detectable in infiltrates but were less numerous, were confined to perivascular locations in the meninges and subpial areas, and did not spread into gray matter (compare Fig. 1Go, D vs E). Also, they were less numerous in infiltrates than Mac-1+ cells (Fig. 1Go, J vs G). Even fewer neutrophils were noted in SJL/J mice examined at earlier time points or with lesser disease severity. Infiltrating leukocytes in SJL/J mice formed perivascular cuffs around PECAM-1+ endothelial cells (Fig. 1GoI). ICAM-1 was also strongly up-regulated in SJL/J, IFN-{gamma}-/-, and IFN-{gamma}R-/- mice (not shown). Thus, lethality was associated with marked neutrophilia in the absence of IFN-{gamma} or its receptor.

Neutrophil infiltration in IFN-{gamma}-/- and IFN-{gamma}R-/- mice was accompanied by extensive demyelination. Luxol Fast Blue was used to stain myelin in Fig. 2Go, A and C. Loss of myelin was localized to areas of spinal cord dominated by PMN infiltration (Fig. 2GoA), so that some sections at other levels showed no evidence of pathology (Fig. 2GoC). As expected, in SJL/J mice, focal zones of demyelination surrounded infiltrated vessels often in subpial infiltrates (not shown). Widespread neutrophil infiltration and demyelination in IFN-{gamma}-/- mice were further confirmed on epoxy sections stained with toluidine blue (Fig. 2GoE). Moreover, electron micrographs revealed the presence of macrophages and PMNs phagocytosing myelin residues (Fig. 2Go, B and D). Neutrophils with the ultrastructure of condensed nuclear chromatin consistent with apoptotic cell death were not observed (Fig. 2Go, B and D). Reactive astrocytes, identified by their hypertrophied appearance, were also prominent in inflammatory and demyelinating regions (not shown). Eosinophils could be identified among the PMNs in knockout mice, but were not detectable in SJL/J mice. Fig. 2GoF shows an electron micrograph of cells with characteristic bilobed nuclear morphology and large ovoid granules in a demyelinated region of spinal cord of IFN-{gamma}-/- mice. These were a minority compared with neutrophils.



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  		FIGURE 2. Extensive demyelination in CNS in mice lacking an IFN-{gamma} response. Sections were derived from spinal cords of mice on day 17 postimmunization. A and C, Luxol Fast Blue staining of paraffin-embedded sections, with (A) and without (C) evidence of infiltration, from the spinal cord of an IFN-{gamma}-/- mouse with grade 5 EAE. E, Toluidine blue staining of an epoxy section of spinal cord of IFN-{gamma}-/- mice shows neutrophil infiltration and associated demyelination. B, D, and F, Electron micrographs of spinal cord from an IFN-{gamma}-/- mouse with EAE showing a neutrophil (arrow) engulfing myelin residues (B) and macrophages (ma) phagocytozing myelin residues (D) among other neutrophils (n) and eosinophils (e) in an infiltrated and demyelinated region. Magnifications: A and C, x25; B, x1050; D, x2000; E, x200; and F, x2400.

 
Macrophage/microglial reactivity

To assess the presence and reactivity of monocytes/macrophages and microglia, we stained for F4/80. Unlike CD11b/Mac-1, which is up-regulated on macrophages/microglia and neutrophils after activation, the expression of the F4/80 glycoprotein is restricted to macrophages and becomes detectable on activated microglia. The F4/80 molecule was not detected in CNS tissues of a naive mouse (not shown), but was strongly up-regulated on macrophages/microglia in the spinal cords of SJL/J mice with EAE (Fig. 1GoH). F4/80+ cells with amoeboid/round morphology were intermingled with infiltrating lymphocytes within inflammatory foci, and some F4/80+-reactive microglia of dendritic morphology were also dispersed in the parenchyma (Fig. 1GoH). Mac-1 staining paralleled that of F4/80 in SJL/J mice. F4/80 staining in CNS of IFN-{gamma}-/- or IFN-{gamma}R-/- mice differed from that in SJL/J mice in both intensity and distribution. The spinal cords of IFN-{gamma}-/- or IFN-{gamma}R-/- mice with acute lethal EAE contained weakly stained F4/80+ cells. These were found girdling the neutrophil-dominated infiltrates (Fig. 1GoK). No F4/80+ cells with dendritic microglia-like morphology were found scattered in the parenchyma of mice deficient in IFN-{gamma} response. As expected, Mac-1 staining followed the same pattern as Gr-1+ neutrophils (compare Fig. 1Go, C vs D), consistent with neutrophil activation. The iNOS immunoreactivity in the CNS of IFN-{gamma}-/- or IFN-{gamma}R-/- mice with EAE was barely detectable, whereas in SJL/J mice (not shown), infiltrates were strongly stained for iNOS during EAE.

CD4+ T cell infiltration and lack of MHC II induction

Disseminated infiltrates in IFN-{gamma}-/- mice contained CD4+ T cells that were interspersed with Gr-1+ cells (Fig. 3GoA, top panels). CD4+ cells had the appearance of lymphocytes with scanty cytoplasm and were discretely stained. By contrast, CD4- cells frequently showed PMN phenotype, having larger cell bodies that were intensively stained for Gr-1. Of note is that Gr-1 staining appeared to extend beyond the cell bodies of PMN, possibly reflecting involvement of extracellular matrix, and formed a densely packed network. Strikingly, no MHC II immunoreactivity was observed in the CNS of IFN-{gamma}- or IFN-{gamma}R-knockout mice with EAE (Fig. 3GoA, bottom panels). However, flow cytometric analysis of cells recovered from the CNS of IFN-{gamma}-/- mice with EAE showed that up-regulation of B7.2 occurred (Fig. 3GoB), and that both CD69 and CD25 were expressed on infiltrating CD4+ T cells (Fig. 3Go, D and E), consistent with recent T cell activation.



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  		FIGURE 3. Infiltration of activated CD4+ T cells to CNS of IFN-{gamma}-/- mice. A, Immunohistochemical staining for CD4 and MHC II on spinal cord sections of IFN-{gamma}-/- and SJL/J mice with grade 4 EAE on day 17 postimmunization. B–E, FACS analysis of cells recovered from the CNS of IFN-{gamma}-/- mice with EAE. B, Dot plot showing expression of B7.2 and Mac-1/CD11b on live-gated cells. C—E, Histograms showing surface staining for CD3 (C), CD69 (D), or CD25 (E) on CD4+ gated cells from the CNS of IFN-{gamma}-/- mice with fulminant EAE. Shaded curves represent controls for nonspecific staining.

 
Up-regulation of MIP-2 and TCA-3 mRNA in CNS

Chemokines influence leukocyte invasion by virtue of their ability to selectively attract and activate subsets of leukocytes. To assess whether the unusual infiltration of the CNS in mice with a defective IFN-{gamma} response reflected a chemokine imbalance, we performed RPA to study chemokine gene expression. As expected, no chemokine mRNA was observed in perfused CNS of naive mice, while multiple chemokine mRNA transcripts were up-regulated during EAE in all mice (Fig. 4Go). In SJL/J mice with MBP/CFA-induced EAE, induction of RANTES and MCP-1 predominated over that of other chemokines, including lymphotactin, eotaxin, IP-10, MIP-1{alpha}, MIP-1ß, and MIP-2 transcripts, and TCA-3 was not detectable (Fig. 4Go, A–C). Similar chemokine expression profiles were observed in spinal cords of SJL/J mice with passively transferred EAE (Fig. 4GoA), suggesting that chemokine gene expression was not influenced by adjuvant. Mice with impaired IFN-{gamma} response, however, displayed a chemokine gene expression profile distinct from that of wild-type mice. MIP-2 and TCA-3 mRNA were markedly up-regulated, while RANTES and MCP-1 mRNA were barely detectable in spinal cords of IFN-{gamma}-/- (Fig. 4GoB) and IFN-{gamma}R-/- mice (Fig. 4GoC) with fulminant EAE. Quantitative analysis revealed a 10-fold increase in MIP-2 transcripts levels in IFN-{gamma}-/- mice compared with that in SJL/J mice with EAE. Conversely, RANTES transcript levels were 20-fold more abundant in SJL/J mice than in IFN-{gamma}-/- mice with EAE.



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  		FIGURE 4. RPA analysis of chemokine mRNA expression in spinal cords of IFN-{gamma}-/-, IFN-{gamma}R-/-, and SJL/J mice with EAE. A, Chemokine mRNA expression in a naive (N) SJL/J mouse or in an SJL/J mouse with active (A) or passive (P) EAE. This is representative of five mice. B, Chemokine gene expression in SJL/J and IFN-{gamma}-/- mice, naive (N) or with EAE. Three other IFN-{gamma}-/- mice with EAE showed the same pattern. C, Chemokine gene expression in IFN-{gamma}R-/- and SJL/J mice, naive (N) or with EAE (E). This is representative of two mice.

 
No Th2 cytokine switch

IFN-{gamma} promotes Th1 responses and suppresses development of Th2 responses. In the absence of IFN-{gamma}, it was possible that Th2 cytokine production would predominate. We therefore examined whether the lack of IFN-{gamma} or IFN-{gamma}R led to a Th2 cytokine response in the CNS.

RPA analysis of RNA from spinal cord of IFN-{gamma}-/- mice with EAE did not detect mRNA transcripts for the Th2 cytokines, IL-4, IL-5, IL-9, IL-10, and/or IL-13 (Fig. 5GoA). The absence of IL-4 mRNA in the CNS of both IFN-{gamma}-/- and SJL/J mice with EAE was further confirmed by a more sensitive RT-PCR assay (Fig. 5GoB). Interestingly, IL-10 mRNA was not expressed in the absence of IFN-{gamma}, although it was detectable in SJL/J mice with EAE (Fig. 5GoB).



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  		FIGURE 5. Cytokine mRNA expression in spinal cords of IFN-{gamma}-/-, IFN-{gamma}R-/-, and SJL/J mice with EAE. A, RPA analysis of cytokine gene expression in SJL/J and IFN-{gamma}-/- mice with (E) and without (N) EAE. B, Duplex RT-PCR analysis for IL-4 mRNA and the housekeeping gene, ß-actin, and RT-PCR for IL-10 mRNA. +control, mouse RNA control (PharMingen) extracted from cells in which housekeeping and specific cytokine genes, including IL-4 and IL-10, were expressed using the baculovirus expression system. C, Levels of IL-12p40 mRNA were detected by RT-PCR and expressed as arbitrary fluorimager units after normalization to ß-actin signals. Bars represent the mean ratio (IL-12p40/ß-actin) in three to four miceper group ± SD in a representative experiment. - and +, Unimmunized (-) and immunized (+) with MBP/CFA. All the immunized mice had developed EAE at the time of analysis, except BALB/c mice.

 
As expected, expression of IFN-{gamma} mRNA was not detected by RPA (Fig. 5Go) or RT-PCR (not shown) in CNS of IFN-{gamma}-/- mice, while it was up-regulated in SJL/J mice with EAE. IFN-{gamma}R-/- mice up-regulated IFN-{gamma} mRNA to lower levels than those in SJL/J mice (not shown). The cytokines IL-2 and IL-12 are also hallmarks of Th1 responses. Comparable levels of IL-2 mRNA transcripts were noted in spinal cords of both IFN-{gamma}-/- and SJL/J mice with EAE (Fig. 5GoA). Consistent with previous studies (36), IL-12p40 mRNA was only weakly detected in spinal cords of unimmunized SJL/J mice, and levels were strongly elevated in EAE (Fig. 5GoC). In contrast, levels of IL-12p40 mRNA in spinal cords of IFN-{gamma}-/- or IFN-{gamma}R-/- mice with EAE were indistinguishable from those in unimmunized BALB/c, IFN-{gamma}-/-, and IFN-{gamma}R-/- mice (Fig. 5GoC). Taken together, these data make it unlikely that a Th2 cytokine bias was responsible for the PMN-dominated CNS pathology seen in mice lacking an IFN-{gamma} response.

IFN-{gamma} disruption in BALB/c enhances T cell response to MBP

Finally, we were interested in how deficiency in IFN-{gamma} response overcame resistance to MBP-induced EAE in BALB/c mice. To test whether disease induction was associated with enhanced T cell reactivity against MBP, we assessed the proliferation of draining LNC from MBP/CFA-primed IFN-{gamma}-/- mice, wild-type BALB/c, and SJL/J controls in a recall response to MBP in vitro. Fig. 6Go shows proliferative responses of LNC 14 days after priming with MBP/CFA. LNC from IFN-{gamma}-/- mice on a BALB/c background that become EAE susceptible proliferated as strongly as SJL/J LNC in response to MBP despite the higher background response that is characteristic of GKO mice (29). Proliferative responses increased as the cell density in the culture increased and consistently paralleled those of SJL/J mice (not shown). BALB/c LNC did not incorporate thymidine in response to MBP to a greater extent than background at any cell density (Fig. 6Go).



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  		FIGURE 6. Enhanced proliferation of LNC from IFN-{gamma}-/- mice in response to MBP. Fourteen days postimmunization with MBP/CFA, LNC (8 x 105/well) from IFN-{gamma}-/-, BALB/c, and SJL/J mice were incubated with or without MBP (50 µg/ml) for 4 days and pulsed with [3H]thymidine during the last 18 h of incubation. Bars show the mean counts per minute of triplicate cultures with SEM. These data are representative of six other experiments. BALB/c LNC responded to PPD (24,223 ± 860 cpm). [3H]thymidine uptake of stimulated LNC from SJL/J or IFN-{gamma}-/- mice was significantly different (p < 0.001) from that of their respective backgrounds, while that of stimulated LNC from BALB/c mice was not (p > 0.05), as determined by one-way ANOVA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Evidence to date has pointed to a detrimental role for IFN-{gamma} in the CNS (35, 42, 43). We have now shown that in the presence of an IFN-{gamma} response, the typical T cell and macrophage chemoattractants, RANTES and MCP-1, and infiltrating mononuclear cells predominate in CNS to produce a nonlethal remitting EAE, while in its absence, MIP-2, TCA-3, and PMN prevail, producing an acute, lethal EAE. Our data thus cast new light on the role of IFN-{gamma} in autoimmune CNS inflammation, as a primary regulator of chemokine profiles. CNS-infiltrating CD4+ T cells are the major sources of local IFN-{gamma} (44), which now appears essential not in the induction but, rather, in the evolution of the autoimmune attack. Our data further suggest that IFN-{gamma} plays an antiproliferative role in EAE-resistant BALB/c mice, such that T cell responses to MBP become detectable in IFN-{gamma}-/- animals. We propose that IFN-{gamma} shapes an autoimmune inflammatory response and exerts a protective role by restricting neutrophil infiltration, activation, and proliferation.

The potential for neutrophil infiltration to the CNS probably exists to mediate host resistance to infection (45). CNS neutrophilia is also associated with acute stroke, traumatic brain injury, fatal hemorrhagic leukoencephalitis, and acute primary progressive forms of EAE and MS (46, 47, 48, 49). These acute reactions often lead to rapid death. Hence, it is important to unravel mechanisms regulating immune cell invasion and activation in inflamed tissues to provide insights into efficient therapeutic intervention. McColl et al. (49) reported that neutrophil depletion inhibits MBP-induced EAE in SJL/J mice and MOG-induced EAE in IFN-{gamma}R-/- mice, suggesting a crucial role for PMN in the pathogenesis of this inflammatory disease. In their study EAE was induced by immunization with MBP or MOG using pertussis toxin as an adjuvant. Pertussis toxin increased blood neutrophilia and vascular permeability (49), making it difficult to dissect mechanisms governing immune cell infiltration of the CNS by IFN-{gamma} and those pertaining to T-cell mediated pathogenesis.

We describe an IFN-{gamma}-regulated neutrophilia in the CNS induced without pertussis toxin or other systemic immunomodulation. IFN-{gamma}-/- or IFN-{gamma}R-/- mice may be predisposed to enhanced myelopoiesis and granulocytosis in the blood and spleen (50, 51), but we found selective recruitment to the CNS. Organs that were not targets of neuroantigen-reactive CD4+ T cells, such as kidneys, were not affected by neutrophilia in IFN-{gamma}-/- mice. The presence of activated CD4+ T cells in the CNS argues for Ag-directed infiltration and focuses attention on the T cells as regulators of immune invasion. IL-3, a growth factor for granulocytes, was up-regulated in the CNS of IFN-{gamma}-/- mice and may have contributed to neutrophil accumulation. Local expansion of neutrophils in CNS may also be dysregulated in the absence of an IFN-{gamma} response. IFN-{gamma} is therefore a key cytokine in coordinating regulation of local autoimmune responses. These data further point to chemokines, MIP-2 and TCA-3, as inducers of neutrophil invasion.

In rodents, MIP-2 acts preferentially on neutrophils and is functionally analogous to human IL-8. Overproduction of MIP-2 using recombinant human adenovirus induced prolonged PMN recruitment to the murine brain (52). MIP-2 is implicated in neutrophil recruitment in bacterial meningitis (53). Anti-MIP-2 treatment reduced neutrophil infiltration and improved survival (53). In the mouse, TCA-3 also acts on neutrophils (54). Our study is the first to show a correlation between MIP-2 and TCA-3 up-regulation and enhanced neutrophilia in a T cell-mediated autoimmune disease.

Down-regulation of MIP-2 expression by IFN-{gamma} might be direct (55) or could be via the action of IL-10. IL-10 was not detectable in the CNS of IFN-{gamma}-/- mice, while it was consistently up-regulated in IFN-{gamma}-intact SJL/J mice. Significantly reduced production of IL-10 was reported from IFN-{gamma}R-/- mice in response to viral Ag (31). Inhibition of IL-10 bioactivity in vivo resulted in a sustained increase in MIP-2 levels (56). IL-10 has been shown to suppress macrophage and neutrophil activities, including cytokine, chemokine, and superoxide production (57). Our finding that mice lacking IFN-{gamma} did not express detectable IL-10 is consistent with neutrophil activation and unopposed MIP-2 up-regulation and supports a disease-modulating role of IL-10 (27, 58, 59).

TNF-{alpha}, superoxide radicals, and NO are implicated as mediators of demyelinating pathology (14, 60). In IFN-{gamma}-/- or IFN-{gamma}R-/- mice, demyelination probably involved reactive oxygen intermediates and/or TNF-{alpha} rather than NO, as very little iNOS immunoreactivity was evidenced in areas of infiltration and demyelination in the spinal cords. IFN-{gamma} appears as a crucial stimulator of iNOS protein expression, as lack of iNOS protein has consistently been reported in IFN-{gamma}-/- or IFN-{gamma}R-/- mice in other infectious or inflammatory diseases (61). Whether reactive oxygen intermediates and/or TNF-{alpha} are the mediators, activated neutrophils were clearly implicated in the demyelination process.

While IFN-{gamma} abrogates MIP-2 production, it promotes RANTES and MCP-1 expression, probably via the synergistic action of TNF-{alpha}. IFN-{gamma} was shown to synergize with TNF-{alpha} to induce human glial cells to increase RANTES production in vitro (62). In mice with intact IFN-{gamma} response, there is a strong up-regulation of RANTES and MCP-1 in the CNS during EAE (4, 20, 63, 64). The fact that such up-regulation did not occur in IFN-{gamma}-/- and IFN-{gamma}R-/- mice suggests that TNF-{alpha}, known to be present in IFN-{gamma}-/- mice (26), is insufficient for response. Our finding that IFN-{gamma} is an important stimulator of RANTES production in EAE is consistent with previous data for other inflammatory diseases in the CNS, such as lymphocytic choriomeningitis (65). That MCP-1 expression was barely detectable in our IFN-{gamma}-/- mice with EAE is also in agreement with the in vitro evidence that IFN-{gamma} up-regulates MCP-1 gene transcription (66).

One of the effects of IFN-{gamma} is to suppress the development of Th2 cytokines. Experimental autoimmune thyroiditis and uveitis, both Th1-mediated diseases, were reported to become biased to Th2-type response in IFN-{gamma}-/- mice (67, 68). Th2-induced pathologies in these diseases were dominated by granulocyte infiltration. Myelin-reactive Th2 cells do not induce EAE in immunocompetent animals, but could transfer disease in immunocompromised mice, and the resulting CNS pathology was again dominated by PMN (13). Similar observations were made in a diabetes model (12). These findings suggest that Th2 cytokines might prevail if EAE were induced in IFN-{gamma} response-deficient mice. Furthermore, in CNS of mice lacking IFN-{gamma} response there was an up-regulation of TCA-3, which, like its human homologue I-309, is a potent chemoattractant for Th2-polarized cells (5). However, we found no evidence for a Th2 cytokine switch in IFN-{gamma}-/- mice during EAE. This argues that PMN-dominated autoimmune pathology is more likely related to lack of IFN-{gamma} regulation of chemokine profiles, rather than to effects of Th2 cytokines.

IL-12 favors the development of a Th1 response and is crucial in the pathogenesis of EAE (27, 69). Systemic administration of anti-IL-12 mAb starting at the time of immunization blocked EAE in IFN-{gamma}-intact or -deficient mice (27, 69). Development of pathogenic autoreactive T cells was abrogated after such systemic IL-12 neutralization or in IL-12-/- mice (27, 69). We did not detect an enhanced induction of IL-12p40 mRNA in the CNS of IFN-{gamma}-/- mice, unlike the case in SJL/J mice. One interpretation of our data could be that disease induction or progression does not require the up-regulation of IL-12 in the local CNS microenvironment, and that the endogenous baseline level of IL-12 is sufficient for the generation of a local immune response.

Interestingly, there was no detectable MHC II staining in infiltrated CNS in IFN-{gamma}-/- or IFN-{gamma}R-/- mice with EAE. This contrasts with IFN-{gamma}-intact mice, in which MHC II is strongly up-regulated on both infiltrating macrophages and reactive glial cells. Although MHC II induction in the CNS was IFN-{gamma} dependent, B7.2 up-regulation did not depend on IFN-{gamma} function. The fact that the T cells in CNS were CD69+ and CD25+ suggests that basal levels of MHC II expression were sufficient for disease induction, although such levels were below the limit of detection by immunohistochemical staining.

It is noteworthy that activated Th1 cells were found in the CNS of IFN-{gamma}-/- mice in the absence of CC chemokines (RANTES, MIP-1{alpha}, and MCP-1) usually associated with a Th1 response. We have shown elsewhere that MIP-1{alpha} is dispensable for Th1 infiltration in EAE,4 and RANTES and MCP-1 may be similarly dispensable. The chemokine IP-10 has been associated with Th1 responses in the CNS (21, 22), and it is possible that despite the low residual levels it promoted Th1 recruitment in the absence of IFN-{gamma}. Other chemokines, not assayed in our experiments, such as Mig, neurotactin, and TCA-4, might also serve as IFN-{gamma}-independent recruitment stimuli for Th1 cells to the CNS. Alternatively, activated T cells might not be dependent on specific chemokines to traffic to the CNS, but may act to induce chemokine production by glial cells. Thus, IFN-{gamma}-deficient T cells, or Th1 in an IFN-{gamma}-unresponsive CNS, might promote a chemokine profile that induces neutrophil infiltration rather than macrophages.

The mice that we studied were on a variety of strain backgrounds. It was not possible to match backgrounds due to the inherent resistance of wild-type mice to MBP-induced EAE. However, the fact that IFN-{gamma}-/- and IFN-{gamma}R-/- mice, on different strain backgrounds showed identical patterns of chemokine expression, infiltration, and disease progression suggests that the influence of IFN-{gamma} response overrides other potential influences. Likewise, patterns of infiltration and cytokine and chemokine production are similar in SJL/J mice with MBP-induced EAE and in C57BL/6 mice with MOG-induced EAE (63) (E. H. Tran, V. Asensio, T. Owens, and I. Campbell, unpublished observations; and M. Hassan-Zahraee, E. H. Tran, and T. Owens, manuscript in preparation). Whether susceptibility and neutrophil pathology could reflect enhanced pathogenic activity of T cells is not supported by the fact that proliferative responses of IFN-{gamma}-/- LN T cells to MBP were never greater than those of SJL/J mice. Furthermore, although there was a high basal proliferative activity of LN T cells, immunization of IFN-{gamma}-/- or IFN-{gamma}R-/- mice with PBS/CFA (Table IGo) or OVA/CFA (not shown) did not induce disease, and maximization of EAE symptoms and penetration in SJL/J mice using adoptive transfers with high T cell numbers does not induce a IFN-{gamma}-/--like pathology. Thus, the simplest and most plausible interpretation of our findings is that the susceptibility of IFN-{gamma}-/- or IFN-{gamma}R-/- mice to EAE can be attributed to the absence of IFN-{gamma} regulation, leading to chemokine imbalance and lethal neutrophil invasion.

Our findings suggest a model for events leading to induction of EAE and MS. IFN-{gamma} is an inhibitor of cellular proliferation, and our data show that IFN-{gamma} deficiency overcame EAE resistance in BALB/c mice by overriding the inability of their LNC to proliferate in response to MBP. For initiation of disease, a suprathreshold frequency of autoantigen-specific CD4+ T cells is required. This entails clonal expansion through proliferation. Strains of animals in which T cell proliferation is curtailed, such as by the action of IFN-{gamma}, will not initiate disease. Our experiments as well as those of Yoshizawa et al. (70) confirm the presence of potentially encephalitogenic T cells in BALB/c. Work by Yoshizawa et al. (70) supports the idea that a high frequency of these T cells, as obtained via in vitro expansion, can transfer disease in BALB/c. Once sufficient numbers of T cells are activated, they migrate to the CNS. Although migration may itself depend on macrophages (71), whether macrophages or neutrophils are finally recruited to the CNS is dependent on whether the CD4+ T cells secrete IFN-{gamma}. This model places IFN-{gamma} in the pivotal role of directing eventual disease outcome via its primary regulation of chemokine secretion. Our model makes no prediction as to the cellular source of chemokines, although CNS glia and leukocytes are plausible candidates. One intriguing possibility that is suggested from this model is that the usual lack of eosinophils in inflamed CNS may result from the autocrine action of CD28-triggered IFN-{gamma} (72). This further predicts that eosinophils in CNS in IFN-{gamma}-intact animals, when present, are not triggered to secrete IFN-{gamma}. Analogous considerations may apply to other potential cell sources of IFN-{gamma}, so that the interplay between IFN-{gamma} secretion and the chemokines it regulates directs leukocyte population dynamics in inflamed tissues such as the CNS.


   Acknowledgments
 
We thank Dr. Alexandre Prat for comments on the manuscript. We are grateful to Lyne Bourbonnière and Maria Caruso for breeding and screening of knockout mice, to Jim Dixon for his expertise in electron microscopy, and to Lily Li and Lixia Zhu for advice on histology and for technical assistance.


   Footnotes
 
1 This work was supported by grants (to T.O.) from the Multiple Sclerosis Society of Canada and the Medical Research Council of Canada and by a Multiple Sclerosis Society of Canada studentship (to E.H.T.). Back

2 Address correspondence and reprint requests to Dr. Trevor Owens, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4. E-mail address: Back

3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; MBP, myelin basic protein; PMN, polymorphonuclear leukocytes; iNOS, inducible NO synthase; MOG, myelin oligodendrocyte glycoprotein; TCA-3, T cell activation gene-3; MCP-1, macrophage chemoattractant protein-1; LNC, lymph node cells; H&E, hematoxylin-eosin; RPA, RNase protection assay; PECAM, platelet endothelial cell adhesion molecule; IP-10, IFN{gamma}-inducible protein-10. Back

4 E. H. Tran, W. A. Kuziel, and T. Owens. Induction of EAE in C57B46 mice deficient in either the chemokine MIP-1{alpha} or its CCR5 receptor. Submitted for publication. Back

Received for publication September 30, 1999. Accepted for publication December 15, 1999.


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 Discussion
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