Dynamic Regulation of α- and β-Chemokine Expression in the Central Nervous System During Mouse Hepatitis Virus-Induced Demyelinating Disease

Thomas E. Lane, Valérie C. Asensio, Naichen Yu, Alyssa D. Paoletti, Iain L. Campbell and Michael J. Buchmeier
J Immunol January 15, 1998, 160 (2) 970-978;

Abstract

Infection of C57BL/6 mice with the V5A13.1 strain of mouse hepatitis virus (MHV-V5A13.1) results in an acute encephalomyelitis and chronic demyelinating disease with features similar to the human demyelinating disease multiple sclerosis. Chemokines are a family of proinflammatory cytokines associated with inflammatory pathology in various diseases. The kinetics and histologic localization of chemokine production in the central nervous system of MHV-infected mice were examined to identify chemokines that contribute to inflammation and demyelination. Transcripts for the chemokines cytokine-response gene-2 (CRG-2), regulated on activation, normal T cell expressed and secreted (RANTES), macrophage-chemoattractant protein-1 and protein-3 (MCP-1, MCP-3), macrophage-inflammatory protein-1β (MIP-1β), and MIP-2 were detected in the brains of MHV-infected mice at 3 days postinfection (p.i.), and these transcripts were increased markedly in brains and spinal cords at day 7 p.i., which coincides with the occurrence of acute viral encephalomyelitis. By day 35 p.i., RANTES, CRG-2, and MIP-1β were detected in brains and spinal cords of mice with chronic demyelination. CRG-2 mRNA expression colocalized with viral RNA and was associated with demyelinating lesions. Astrocytes were the predominant cell type expressing CRG-2 mRNA. These observations suggest a role for chemokines, notably CRG-2, in the initiation and maintenance of an inflammatory response following infection with MHV, which is important in contributing to demyelination.

Infection of mice with neurotropic strains of mouse hepatitis virus (MHV),3 a member of the coronaviridae family, results in a rapidly progressive encephalomyelitis that may culminate in death or survival. Animals that survive often develop a paralyzing demyelinating disease with clinical and pathologic similarities to the human demyelinating disease MS (1, 2, 3). Whether demyelination is the result of persistent viral infection of the CNS or due to the loss of self-tolerance and the concomitant development of sensitization to neuroantigen(s) is not clear. MHV-induced acute demyelination has been attributed to direct lysis of virally infected oligodendrocytes (4, 5). However, current evidence indicates that the host cellular immune response is a key factor in contributing to the demyelinating process. Infection of SCID mice or γ-irradiated mice with MHV does not result in demyelination, even though these animals exhibit high viral titers in the CNS (2, 6). Furthermore, adoptive transfer of spleen cells to SCID or γ-irradiated animals results in active demyelination (2, 6). Taken together, these studies suggest an immunologic basis for demyelination in MHV-infected mice.

Maintenance of a chronic inflammatory response appears to be an important aspect contributing to demyelination in MHV-infected spinal cords. A prominent feature of MHV-induced demyelination is the presence of inflammatory mononuclear cell infiltrates, including T lymphocytes and monocytes associated with demyelinating lesions (4). Sun et al. (7) have demonstrated recently the expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 by astrocytes in the spinal cords of chronically infected mice. These cytokines may act individually or in concert to recruit cells, such as T lymphocytes and monocytes, to sites of viral infection, which then participate in demyelination (7). However, the soluble signals that regulate CNS invasion by inflammatory cells are likely to consist of multiple factors that have yet to be fully defined, and remain a key question in understanding the pathology of chronic demyelination in MHV-infected mice.

The chemokines represent a family of low m.w., proinflammatory cytokines that are divided into two major subfamilies based on structural and functional criteria (8, 9). The α-family is structurally characterized by a conserved C-X-C motif in the amino terminus of the protein, with the X representing an amino acid separating two cysteine residues, while the β-chemokine family contains two cysteines that are adjacent, i.e., C-C (8, 9). Recently, a third chemokine, lymphotactin, has been identified (10). The individual subfamilies of chemokines have been shown to selectively attract distinct leukocyte populations during periods of inflammation (8). In general, the α-chemokines function in attracting neutrophils, yet have limited effect on T lymphocytes or monocytes. However, there are exceptions to this rule, as CRG-2 (the murine homologue of the human chemokine IP-10) has been demonstrated to be chemotactic for activated T cells and monocytes (11). The β-chemokines predominantly attract T cells, monocytes, and macrophages, but have only limited effect on neutrophils (8). Lymphotactin appears to be strictly lymphocyte specific (10). Recent work has shown that chemokine production is associated with inflammatory pathology in CNS diseases, including models of demyelination such as EAE (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). We have investigated production of chemokine mRNA transcripts in the CNS of mice suffering from both acute and chronic MHV infection to more fully define factors that control inflammation and demyelination in this model.

Materials and Methods

Virus and mice

The MHV strain V5A13.1 was derived from the wild-type MHV-4, as previously described (22). Infection of susceptible strains of mice with the neuroattenuated MHV-V5A13.1 results in a chronic demyelinating disease in susceptible animals (22). Viral titers from brains of infected animals were determined by plaque assay on the DBT astrocytoma cell line, as previously described (22, 23, 24). Age-matched (5–7 wk) male C57BL/6 mice (H-2b background) were used for all studies described. Following anesthetization by inhalation of methoxyflurane (Pitman-Moore, Washington Crossing, NJ), mice were injected intercranially with 30 μl of 10 PFU of MHV-V5A13.1 suspended in sterile saline. Control animals were injected with sterile saline alone. Animals were killed by methoxyflurane anesthesia at days 2, 3, 7, 15, 21, and 35 p.i., and brains and spinal cords were removed. One-half of each brain of all killed animals was used for plaque assay to determine viral burden. The remaining half brain as well as spinal cords were either fixed in 10% normal buffered Formalin for paraffin embedding, frozen in O.C.T. compound (Miles, Elkhart, IN), or stored at −70°C for RNA isolation.

Cell culture and viral infection

Primary cultures of cerebral cortical astrocytes were prepared from newborn C57BL/6 mice (1–2 days old). Forebrains were removed aseptically from the skull, the meninges were excised carefully under a dissecting microscope, and the neocortex was dissected. The cells were dissociated by being passed through needles of decreasing gauge sizes (16, 19, and 25 gauge) two or three times with a 10-ml syringe. Trypsin was not used for tissue dissociation. The cells were seeded at a density of 105 cells/cm2 on six-well plates in DMEM containing 10% FCS and 25 mM glucose in a final volume of 2 ml/well and incubated at 37°C in an atmosphere containing 5% CO2 at 95% humidity. The culture medium was renewed 3 to 4 days after seeding and subsequently twice per week. These conditions yielded astrocyte cultures containing >90% GFAP-immunoreactive cells and <1% Mac-1-positive cells. Confluent monolayers of cells were infected with increasing concentrations of MHV-V5A13.1: 0, 0.1, 0.5, 1, 5, 10, and 50 PFU/ml. In addition, 10 and 50 PFU of virus was UV inactivated and added to separate monolayers. Virus was allowed to adsorb for 1 h, at which time monolayers were washed and replaced with 2 ml fresh medium. Infected monolayers were incubated for 24 h, at which time total RNA was extracted using TRIzol reagent (Life Technologies) and 10 μg used for RNase protection assay (RPA) (described below).

RNase protection assay

Total RNA was extracted from brain and spinal cords using the TRIzol reagent, as previously described (24). For RPA, a multiprobe set was used that detected the following chemokine transcripts: α-chemokines; MIP-2 and CRG-2; β-chemokines; C10, RANTES, MCP-1, MCP-3, MIP-1α, MIP-1β, and T cell activation-3; and lymphotactin. A probe for L32 was included in the probe set to verify consistency in RNA loading and assay performance. Details on the construction and performance of the multiprobe chemokine RPA set have been described recently (25). RPA analysis was performed on 10 μg total RNA using a previously described protocol (25, 26, 27). Following separation by PAGE, protected 32P-labeled probe fragments were visualized by film autoradiography. Controls included the probe set hybridized to transfer RNA only and to transfer RNA plus an equimolar pool of synthetic-sense RNAs complementary to the probe set. For quantification, autoradiographs were scanned (Scanjet 4C/T; Hewlett Packard, San Jose, CA) and band density was assessed with National Institutes of Health image 1.57 software.

Reverse-transcription PCR

The antisense riboprobe used to detect CRG-2 mRNA was derived by RT-PCR amplification of cDNA generated from total RNA isolated from the brain of an MHV-V5A13.1-infected mouse at day 7 p.i. (24). Oligonucleotide primers for CRG-2 amplification were as follows: 5′-CAG CAC CAT GAA CCC AAG TGC and 5′-GCT GGT CAC CTT TCA GAA GAC C. PCR amplification for the oligonucleotide primers was performed using an automated Perkin-Elmer (Norwalk, CT) model 480 DNA thermocycler with the following profile: step 1, initial denaturation at 94°C for 45 s; step 2, annealing at 60°C for 45 s; and step 3, extension at 72°C for 2 min. Steps 1 to 3 were repeated 34 times for a total of 35 cycles. A final extension at 72°C of 7 min was performed. The PCR amplicon was subjected to gel electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining (0.5 μg/ml). For CRG-2, the expected 450-bp fragment was cut out of the gel and extracted using the Gene Clean II system (Bio 101, San Diego, CA). This fragment was cloned into the pCR Script SK+ vector (Stratagene, La Jolla, CA), and sequence analysis identified ≥95% nucleotide identity with mouse CRG-2 (28).

In situ hybridization

The protocol for in situ hybridization of brain and spinal cord sections as well as the description of the riboprobe for MHV has been described previously (24). The CRG-2 antisense riboprobe was generated by linearization with the NotI restriction enzyme, while the control sense probe was generated using the EcoRI restriction enzyme. The [35S]UTP-radiolabeled RNA probes were derived by in vitro transcription reaction with an RNA transcription kit (Stratagene). Upon completion of the in situ procedure, the slides were dehydrated and dried. Next, the slides were dipped in a Kodak NTB2 nuclear emulsion at 42°C and exposed at 4°C for 2 to 4 wk in a desiccator. The slides were developed and fixed with Kodak D-19 developer and fixer, counterstained with hematoxylin and eosin Y solutions, dehydrated, and mounted.

Immunohistochemistry

Abs used for immunohistochemical detection of cellular Ags are as follows (diluted in PBS): anti-bovine GFAP (rabbit polyclonal; Dakopatts, Carpinteria, CA), 1/1000; anti-CD4 (rat mAb L3T4; PharMingen), 1/100; anti-CD8 (rat mAb Ly-2 and Ly-3; PharMingen, San Diego, CA), 1/200; and anti-Mac-1 (rat mAb M170). In all cases, a biotinylated secondary Ab was used (1/300; Vector Laboratories, Burlingame, CA). Staining for GFAP was performed on brain and spinal cord sections fixed in 10% normal buffered Formalin and embedded in paraffin according to previously described protocols (24, 27). Immunohistochemical analyses for CD4, CD8, and Mac-1 Ags were performed on frozen sections fixed in acetone at −20°C. The ABC Elite (Vector Laboratories) staining system was used according to manufacturer’s instructions, and either VIP or diaminobenzidine was used as a chromagen. All slides were counterstained with hematoxylin.

Combined immunohistochemistry and in situ hybridization

For studies designed to colocalize cellular Ags with in situ signals for CRG-2 mRNA, slight modifications in the procedures were implemented. In all cases, immunohistochemical analysis preceded in situ hybridization. PBS used for dilution of Abs as well as in washing steps was Diethyl pyrocarbonate treated to reduce RNase contamination and loss of in situ signal. For studies done on frozen sections, the slides were dried overnight in a desiccator and fixed in 4% paraformaldehyde for 10 min at 4°C before beginning the immunohistochemistry/in situ procedure. Following application of diaminobenzidine, slides were washed twice in PBS and then prehybridized in hybridization buffer for 1 h at 42°C. Following this incubation, the linearized 35S-labeled riboprobe was added to the sections and the standard in situ hybridization procedure was followed. Upon development, the slides were counterstained in hematoxylin only, then dehydrated and mounted.

Results

MHV infection and demyelination

Infection of C57BL/6 mice with MHV-V5A13.1 resulted in an acute viral encephalomyelitis with approximately 10% mortality. The surviving animals developed the clinical characteristics of MHV-induced demyelination, e.g., awkward gait and hind-limb paralysis between days 15 and 35 p.i. Virus could not be isolated from the majority of infected mice by day 15 p.i., as determined by plaque assay (limit of detection ∼100 PFU/g tissue). Examination of brains and spinal cords of MHV-infected animals at day 35 p.i. revealed extensive demyelination with persistent viral RNA associated with white matter tracts (Fig. 1⇓). CD4+ and CD8+ T cell subsets as well as Mac-1-positive cells were closely associated with white matter lesions in spinal cords of mice (Fig. 2⇓). A summary of these clinical and pathologic observations is shown in Table I⇓.

FIGURE 1.
FIGURE 1.

Persistent viral RNA and demyelination. Spinal cord section from an infected mouse showing evidence of viral RNA associated with demyelination. MHV RNA in white matter tract of spinal cord at day 35 p.i. is detected by in situ hybridization. Virally infected cells are indicated by overlaying silver grains. Original magnification, ×400.

FIGURE 2.
FIGURE 2.

Cellular infiltrate associated with plaque lesion in spinal cord of MHV-infected mice. Sequential frozen sections from the spinal cord of an infected C57BL/6 mouse at day 15 p.i. were subjected to immunohistochemical staining for the designated cellular Ags. Note the localization of CD4+-, CD8+-, and Mac-1-positive cells around a demyelinating plaque lesion. Control panel represents nonspecific Ab. Original magnification, ×400.

View this table:
Table I.

Summary of clinical and pathological features in MHV infection

CNS chemokine expression following MHV infection

In attempt to identify chemokines expressed temporally during early and late stages of MHV infection, RNA was extracted from the brains and spinal cords of infected animals and subjected to RPA using a probe set designed to detect 10 different chemokine transcripts (25). As shown in Figure 3⇓, chemokine mRNA transcripts were detected at various times following infection with MHV. At day 3 p.i., a transcript for the α-chemokine CRG-2 was expressed abundantly in the brain. In addition to CRG-2, transcripts for the β-chemokines MCP-1, MCP-3, MIP-2, and RANTES were also detected, although at much lower levels. Very low chemokine expression was observed at day 3 in the spinal cord. A dramatic increase in mRNA expression of the β-chemokines MIP-1β, MCP-1, MCP-3, and RANTES as well as CRG-2 was observed in the brains and spinal cords of animals by day 7 p.i., which coincided with the increased numbers of inflammatory cell in the brain and spinal cord at this time. By day 35 p.i., surviving animals had cleared infectious virus (but not viral RNA), and RANTES, CRG-2, and MIP-1β mRNAs were the predominant chemokines in brains and spinal cords.

FIGURE 3.
FIGURE 3.

A, RPA showing kinetics of induction of chemokine mRNA following intercranial injection of MHV. Total RNA was extracted from the brains and spinal cords of C57BL/6 mice at the indicated time points, and 10 μg hybridized with a 32P-labeled antisense probe set designed to detect 10 different chemokine transcripts. A probe for L32 was included in the probe set to verify consistency in RNA loading and assay performance. Each lane indicates either brain or spinal cord from an individual mouse. A (+) indicates a mouse infected with MHV-V5A13.1, while a (−) indicates a sham-infected control animal. After separation by PAGE, protected probe fragments were visualized by autoradiography. A sample consisting of a set of sense RNAs complementary to the probe set for use in standardization of fragment size and assay integrity was included and shown on the right-hand margin of the autorad. These sense RNAs contain cloning sequences, and consequently run slightly higher than protected fragments from brain and spinal cord. Up-regulated chemokines are indicated in the left-hand margin. Controls included RNA from the brains and spinal cords of sham-infected C57BL/6 mice. B, Quantitative analysis of chemokines expressed in brains of infected mice. Densitometric analysis of each lane representing a brain sample from an individual mouse was performed on the scanned autoradiograph (A) using National Institutes of Health image software. The levels are based on arbitrary units that allow comparison of chemokine mRNA transcript levels at 3, 7, and 35 days postinfection with virus. Data are presented as the average ± SE of the mean of each chemokine signal from individual mice at each time point. The sample sizes are as follows: day 3, n = 2; day 7, n = 4; day 9, n = 9; sham control day 3, n = 1; and sham control day 7, n = 2. C, Quantitative analysis of chemokines expressed in spinal cords in infected mice. Densitometric analysis of each lane representing a spinal cord sample from an individual mouse was performed on the scanned autoradiograph (A) using National Institutes of Health image software. Data are presented as described in B figure legend. The sample sizes are as follows: day 3, n = 2; day 7, n = 3; day 35, n = 5; and sham control day 7, n = 2.

To provide more quantitative interpretation of these data, the autoradiograph in Figure 3⇑ was scanned, and the density of individual chemokine bands was measured and the data presented as a ratio of chemokine band intensity to the L32 control. Brains and spinal cords from mice at each time point were analyzed separately, and the data presented in Figure 3⇑, B and C, represent the average signal intensity for each chemokine following infection. There was an approximately 80-fold increase in CRG-2 mRNA as compared with sham-infected controls at day 3 p.i. in the brain, while there was approximately a 25-fold increase in CRG-2 expression in the spinal cord (Fig. 3⇑, B and C). A 20-fold increase in MCP-1 was observed in the brain at this time, followed by increases of approximately 15-fold for MCP-3 and MIP-1β and an 8-fold increase in RANTES expression. By day 7 p.i., the average level of CRG-2 in the brain dropped slightly as compared with day 3, although it remained the predominant chemokine transcript to be expressed in the brain. Compared with day 3, there was a dramatic increase in chemokine mRNAs, with 2- to 4-fold increases in MCP-1, MCP-3, and MIP-1β in the brain. RANTES expression in the brain was approximately 4-fold higher at day 7 as compared with day 3. An increase in chemokine mRNAs was observed in the spinal cord of MHV-infected mice, with an approximate 4-fold increase in CRG-2 expression as compared with day 3, while there was a 3- to 8-fold increase in the expression of MCP-1, MIP-1β, MCP-3, and RANTES. By day 35 p.i., a drop in levels of chemokine mRNAs occurred in both brains and spinal cords. RANTES, CRG-2, and MIP-1β are the predominant chemokines expressed in these tissues at this time point. RANTES was expressed at slightly higher levels than CRG-2, which, in turn, was expressed at higher levels as compared with MIP-1β.

CRG-2 expression and MHV infection

We examined the expression of CRG-2 in more detail because this particular chemokine was expressed both early (day 2) and late (day 35) following infection with MHV. In situ hybridization analysis using antisense probes for CRG-2 and MHV was performed on sequential sagittal sections of brain and spinal cord to determine the spatial relationship between viral RNA and CRG-2 expression. The data in Figure 4⇓ demonstrate the colocalization of viral RNA with CRG-2 mRNA at days 2 and 7. By day 21 p.i., both viral RNA and CRG-2 mRNA could be detected in the brain; however, there was not the strict colocalization as was observed at days 2 and 7. Examination of spinal cords at day 35 p.i. revealed expression of CRG-2 mRNA in white matter tracts associated with areas of demyelination (Fig. 5⇓). Positive cells were found predominantly on the edge of demyelinating lesions.

FIGURE 4.
FIGURE 4.

MHV RNA and CRG-2 mRNA expression in the brain. In situ hybridization showing distribution of viral RNA and CRG-2 mRNA in brains of MHV-infected mice. Three sequential sagittal sections of paraffin-embedded brain sections from infected mice at indicated time points were probed with 35S-labeled antisense riboprobes specific for either MHV or CRG-2. As a control, a sense probe for CRG-2 was included. Signal was detected by autoradiography following a 5-day exposure to film. The probes used for each section are indicated. Note the strict colocalization of CRG-2 mRNA with viral RNA at days 2 and 7 postinfection.

FIGURE 5.
FIGURE 5.

CRG-2 mRNA is expressed in areas of demyelination. CRG-2 mRNA was detected by in situ hybridization in white matter tracts of demyelinating spinal cords at day 35 p.i. A, CRG-2-positive cells (arrows) adjacent to demyelinating lesions. Original magnification ×400. B, Spinal cord section in which the sense control probe for CRG-2 was used. No positive cells were detected.

Identification of CRG-2-producing cells

Resident glia that have been shown to express CRG-2 include microglia and astrocytes (13, 28, 29). Double labeling using an antisense riboprobe for CRG-2 mRNA and a polyclonal Ab to either GFAP (specific for astrocytes) or Mac-1 (recognizes microglia/macrophage) was performed to determine whether these cell types were responsible for production for CRG-2 mRNA following infection with MHV. These experiments revealed that during acute stage of infection, e.g., day 7, both astrocytes and Mac-1-positive cells were found to express CRG-2 mRNA; however, astrocytes were the predominant cell type expressing this chemokine transcript (Fig. 6⇓). During early and late stage of disease (day 3 and day 35), only astrocytes were found to express CRG-2 in both brain and spinal cord.

FIGURE 6.
FIGURE 6.

Astrocytes and Mac-1-positive cells express CRG-2 mRNA in the CNS of MHV-infected mice. Combined immunohistochemistry for either GFAP or Mac-1 and in situ hybridization for CRG-2 mRNA were performed on the brain of a mouse following infection. Left panel, Astrocytes and their processes are stained brown and are identified as being positive for CRG-2 mRNA expression at day 7 p.i. by overlaying silver grains (arrows). Right panel, Mac-1-positive cells, stained brown, expressing CRG-2 at day 7 p.i. are identified as being positive by overlaying silver grains from in situ procedure (arrow). Original magnification, ×400.

Astrocyte expression of chemokines and MHV infection

The in vivo data indicated that astrocytes were the predominant cell type expressing CRG-2 mRNA at both early and late stages of infection. We wished to extend these findings and study the chemokine transcript profile by primary astrocytes following in vitro infection with MHV-V5A13.1. Primary astrocytes were cultured from the brains of 1- to 2-day-old C57BL/6 mice and infected with increasing concentrations of virus. In addition, separate monolayers were infected with UV-inactivated virus to determine whether MHV replication was required for expression of chemokines. Total RNA was isolated from the cell monolayers following a 24-h incubation and subjected to RPA with the chemokine probe set. The results presented in Figure 7⇓A indicate that virally infected astrocytes expressed transcripts for MIP-2, MCP-3, MIP-1β, MCP-1, and CRG-2. Low level expression of MIP-1β was evident even in control monolayer cultures, suggesting that isolation of cells may have resulted in slight activation of the cells. These results are interesting in that the chemokine profile of MHV-infected astrocytes is similar to what was observed in MHV-infected animals, with CRG-2, MCP-1, MIP-1β, MCP-3, and MIP-2 being expressed both in vivo and in vitro. A notable difference was that RANTES was not expressed by MHV-infected astrocytes in vitro, while it was detected in the brains and spinal cords of MHV-infected mice. Quantitation of the signal intensities reveal that at MHV concentrations lower than 10 PFU/ml, MIP-1β was the prominent chemokine expressed (Fig. 7⇓B). Increasing MHV to 50 PFU/ml resulted in a shift in expression, with CRG-2 being expressed at the highest levels, with approximately a sixfold increase over medium control, followed by MCP-1, MIP-1β, MIP-2, and MCP-3. UV inactivation of virus resulted in approximately a two- to fourfold decrease in expression of all chemokine transcripts. However, chemokine expression was not reduced to the levels observed in cells incubated with medium only, suggesting that astrocyte expression of chemokine genes was not entirely dependent upon MHV replication.

FIGURE 7.
FIGURE 7.

A, Chemokine transcripts from primary astrocytes infected with MHV. Primary astrocytes were cultured from the brains of C57BL/6 neonatal mice and infected with various doses of MHV-V5A13.1 (indicated above lanes). Total RNA was collected from the monolayers after 24-h incubation and subjected to RPA with the chemokine probe set. UV-inactivated virus is indicated. Up-regulated chemokine transcripts are indicated in the left-hand margin. B, Quantitation of chemokine transcripts from MHV-infected astrocyte monolayers. Densitometric analysis was performed on the scanned autoradiograph from A using National Institutes of Health image software. The levels are based on arbitrary units that allow comparison of chemokine mRNA transcript levels following the 24-h incubation period.

Discussion

Host factors that function to recruit cells into areas of inflammation are of central importance in the initiation and maintenance of the inflammatory process in the CNS (8, 12, 30). A variety of soluble mediators of chemoattractants have been described, including the anaphylotoxin C5a, leukotriene B4, and cytokines such as TNF-α and IL-1β (31, 32, 33). Recent studies have defined a unique family of structurally related small, chemotactic cytokines termed chemokines. The chemokines have been shown to direct the specific migration of distinct leukocyte populations during periods of inflammation in wide variety of disease processes including trauma (16, 34, 35), autoimmune disorders (12, 13, 14), and microbial infection, including viral disease (36, 37, 38, 39, 40, 41). Cook and colleagues (42) demonstrated that homozygous MIP-1α knockout mice were resistant to Coxsackievirus-induced myocarditis, suggesting an important role for this chemokine in mediating virus-induced inflammation in vivo. Sasseville et al. (43) have shown elevated expression of the β-chemokines MCP-3, MIP-1α and MIP-1β, RANTES, and the α-chemokine CRG-2/IP-10 in SIV-induced encephalitis, suggesting that these chemokines could be involved in leukocyte recruitment to the brain in SIV-infected rhesus macaque monkeys. These observations lend support to the idea that chemokines play an essential role in the development of inflammation in response to viral infection.

Production of chemokines in EAE has also been suggested to play an important role in both the acute as well as chronic stages of disease. Multiple chemokines have been shown to be up-regulated preceding and during the acute stage of EAE (14, 16, 17). Glabinski et al. (13) recently have shown coordinate up-regulation of MCP-1, MIP-1α, KC, RANTES, and CRG-2/IP-10 in the brain and spinal cord during spontaneous relapses of chronic EAE. These data suggest that during the course of this autoimmune demyelinating disease, there is strict regulation of chemokine expression, which in turn regulates CNS inflammation and disease process.

In this study, we have analyzed the time course and cellular source of chemokine gene expression in an animal model of virus-induced demyelination. Both α- and β-chemokines were expressed during subacute, acute, and chronic stages of disease in the brains and spinal cords of MHV-V5A13.1-infected animals. As early as day 2, the α-chemokine CRG-2 was expressed in the brain, and a limited number of inflammatory cells was present. However, by day 7 p.i., the animals were suffering from an acute viral encephalomyelitis characterized by widespread dissemination of virus throughout the brain as well as spinal cord, and a massive inflammatory response that included T cells (both CD4+ and CD8+ T cell) and macrophages. This particular time point also reflected an enhanced expression of mRNA transcripts for the β-chemokines MCP-1, MCP-3, MIP-1β, and RANTES, in addition to CRG-2. By day 35 p.i., the animals had cleared the majority of infectious virus, yet viral RNA persisted in white matter tracts of the brain and spinal cord. Clinically, the animals exhibited awkward mobility and, in more extreme cases, hind-limb paralysis. Histologic examination of the brain and spinal cords revealed extensive white matter demyelination. Although present in limited numbers, inflammatory cells were observed in the brains and spinal cords of these animals. Transcripts for RANTES, CRG-2, and MIP-1β mRNA were the predominant chemokines expressed at this time. These studies indicated that chemokine expression was regulated during different phases of MHV-induced disease.

A correlation between chemokine mRNA transcripts and protein expression was not examined in this study. Therefore, while the data presented in this study convincingly show chemokine gene expression in the CNS following MHV infection, it remains a possibility that certain chemokine transcripts may not be expressed at the protein level. However, the fact that chronic infiltration by lymphocytes into the CNS of infected animals is observed suggests that chemoattractants are being expressed and influencing inflammation. Current studies in our laboratory are investigating chemokine protein expression in the CNS of MHV-infected mice during disease.

The expression of CRG-2 was examined in more detail because 1) it was expressed at all stages of MHV infection examined, 2) CRG-2 mRNA strictly colocalized with areas of viral RNA in brain at days 2 and 7 p.i., and 3) CRG-2 mRNA is associated topographically with areas of demyelination. Together, these observations suggest a role for this particular chemokine in MHV-induced CNS disease. In addition, CRG-2 is the predominant chemokine expressed in SIV encephalitis (43) and lymphocytic choriomeningitis (25). Thus, prominent expression in diverse viral infection of the CNS suggests an important role in the CNS host response to viral infection. The in vivo function of CRG-2 during MHV-induced CNS disease is not presently known. CRG-2 is a potent in vitro chemoattractant for activated T lymphocytes as well as monocytes (11). Recent studies have shown that CRG-2 is expressed during the acute and chronic stages of EAE, suggesting that it contributes to the disease process (13, 29). This is supported by observations that have shown that blocking CRG-2 expression by intrathecal administration of antisense oligonucleotides resulted in a significant decrease in the severity of EAE (44). A single chemokine may exert an essential, nonredundant role in the development of inflammatory disease, as evidenced by studies by Karpus et al. (45), which showed that anti-MIP-1α Abs delivered in vivo blocked EAE. Additional studies have demonstrated that in vivo neutralization of select chemokines has a profound influence on the recruitment of T lymphocytes and monocytes to sites of delayed hypersensitivity reactions, pulmonary granulomas, and interstitial pneumonia and fibrosis (46, 47, 48, 49).

The cytokine IFN-γ has been shown to be a potent agonist in activating both microglia and astrocytes to express and secrete CRG-2 (44). Intravenous injection of mice with IFN-γ results in tissue-specific expression of CRG-2 (36, 50). Although IFN-γ mRNA is present during the acute phase of infection, i.e., day 7 p.i., we were not able to detect any IFN-γ at either day 3 or day 35 p.i., yet CRG-2 mRNA was readily detectable at these time points, suggesting that IFN-γ is not strictly required for CRG-2 expression. This is consistent with a recent study by Amichay and colleagues (36), which demonstrated that induction of CRG-2 following infection with various microbes including vaccinia virus was not completely dependent on IFN-γ expression. In addition, a recent study by Asensio and Campbell (25) has demonstrated that CRG-2 is expressed in the brains of IFN-γ knockout mice during lymphocytic choriomeningitis. The majority of CRG-2-positive astrocytes were not infected with MHV, as determined by in situ hybridization experiments, suggesting that astrocyte expression of CRG-2 may be a secondary event preceded by either cytokine/chemokine production by MHV-infected cells that then function in both an autocrine and/or paracrine manner to activate neighboring, uninfected astrocytes to express CRG-2.

The fact that CRG-2 mRNA is expressed predominantly as early as day 2 in the absence of a prominent inflammatory infiltrate suggests that MHV infection alone is sufficient to trigger CRG-2 expression by resident astrocytes. Therefore, it is reasonable to suggest that CRG-2 is a key signal in initiating the inflammatory response to MHV infection of the CNS. By day 7 p.i., there is a dramatic up-regulation in mRNAs for the chemokines MCP-1, MCP-3, MIP-1β, and RANTES as well as CRG-2. Furthermore, in infections with a related MHV strain, increased expression of the cytokines IFN-γ and TNF-α was also present at this time, which may potentiate expression of certain chemokines (24, 27). The increased expression of cytokines and chemokines observed is most likely a reflection of the massive influx of CD4+ and CD8+ T cells and macrophages in response to the high viral burden at this stage of the infection. Infiltrating mononuclear cells, in addition to resident glia, are most likely responsible for the production of these chemokines.

The β-chemokines appear to be the predominant chemokine family expressed in the CNS during inflammatory disease, including EAE and SIV encephalitis (13, 14, 43). We have shown that β-chemokines are expressed predominantly during the acute phase response to MHV infection of the CNS. However, during both early (2 days) and late (35 days), the α-chemokine CRG-2 is a predominant chemokine expressed in the brain and spinal cord. The significance of these observations is not clear. The β-chemokines tend to target T lymphocytes and macrophages, which are the major immune cell participants in various autoimmune disorders as well as in microbial infections. In contrast, the α-chemokines generally attract neutrophils during inflammation. However, CRG-2 is an α-chemokine that has been shown to be chemotactic for T lymphocytes and monocytes (11). Therefore, chronic expression of CRG-2 in the CNS of MHV-infected animals may aid in attracting T cells and macrophages to sites of viral persistence.

Studies examining chemokines and CNS inflammatory disease have demonstrated that astrocytes are a primary source of chemokines. The in vivo results presented in this study indicate that astrocytes were the predominant cell type to express CRG-2 mRNA during the subacute, acute, and chronic phases of MHV infection. Mac-1-positive cells were also found to express CRG-2 mRNA at day 7 p.i., which represented the height of the inflammatory response, with CD4+ and CD8+ T cells present in the brain and spinal cord as well as increased expression of chemokines and cytokines. In such an environment, the presence of these activating factors appears to be sufficient to stimulate CRG-2 expression by Mac-1-positive cells. The majority of such cells most likely represent microglia and macrophages; however, other cell types, such as neutrophils and NK cells, also express the Mac-1 Ag. Previous work by Williamson et al. (51) has demonstrated that NK cells are present in the brain at day 7 following infection with MHV; therefore, it is possible that some CRG-2-positive cells are NK cells. However, the majority of Mac-1-positive cells had the morphology of macrophage and/or microglia, suggesting that these cell types were responsible for CRG-2 expression.

In vitro studies with astrocytes treated with MHV indicate that a similar chemokine profile exists as compared with the in vivo chemokine profile obtained from MHV-infected mice. The chemokines CRG-2, MCP-3, MIP-1β, MCP-1, and MIP-2 were expressed in MHV-infected brains and spinal cords as well as MHV-infected astrocytes. In contrast, RANTES is expressed in MHV-infected animals and not in astrocytes exposed to MHV. At the highest concentration of MHV (50 PFU/ml), CRG-2 was the predominant chemokine expressed. Similarly, CRG-2 was the predominant chemokine observed in MHV-infected brains and spinal cords during the acute phase of the disease and was expressed at day 35 in brains and spinal cords. In both circumstances, astrocytes were the primary cell type responsible for CRG-2 expression. UV inactivation of virus resulted in a marked decrease in chemokine gene expression, suggesting that active viral replication contributes to expression of chemokine genes. However, there was not complete reduction in expression, indicating that chemokine expression was not entirely dependent upon MHV replication. Similar results were reported in a recent study by Vanguri and Farber (49), which examined the relationship between chemokine expression by astrocytes and NDV. Expression of CRG-2 by astrocytes was dependent on the concentration of NDV. In addition, UV-inactivated NDV was also able to induce expression of CRG-2 by astrocytes. Together, these results suggest that astrocytes may be exquisitely sensitive to exposure to infectious agents such as viruses, and as a result are activated to send an emergency signal, i.e., chemokines that result in the recruitment of effector cells to sites of infection. An unresolved question regarding MHV-induced chronic demyelination is the state of the persistent viral RNA present in the CNS and what role it has in contributing to chronic inflammation. Our results indicate chemokine expression is not dependent upon MHV replication. These results may be extended to suggest that even if persistent virus is present in the CNS, yet is unable to replicate, i.e., it is defective/translationally inactive, it may still be stimulating chemokine expression, which in turn is contributing to chronic demyelinating disease by attracting effector cells into the CNS to sites of viral RNA.

The results presented in this study clearly show that viral infection of the CNS results in an orchestrated cascade of chemokine gene expression. These results indicate that the regulation of chemokine expression contributes to the initiation and maintenance of the chronic inflammatory response that is observed in MHV-infected mice. Production of chemokines, notably CRG-2, by resident astrocytes may aid in attracting T cells and microglia/macrophage to areas in which MHV is present, and these cells participate in the demyelination process through, as of yet, undiscovered mechanisms.

Acknowledgments

We thank Jim Johnston for help in manuscript preparation and Howard Fox for technical advice and discussion.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants AI 25913 and MH 19185 to M.J.B. I.L.C. is supported by National Institutes of Health Grants MH 50426 and MH 47680. T.E.L. is a senior postdoctoral fellow of National Multiple Sclerosis Society. V.C.A. was supported by IPSEN Foundation (Paris, France) and AFFDU Foundation (Paris, France). This is TSRI publication 10775-NP.

  • ↵2 Address correspondence and reprint requests to Dr. Thomas E. Lane, Department of Neuropharmacology CVN8, The Scripps Research Institute, La Jolla, CA 90034. E-mail address: tlane{at}scripps.edu

  • 3 Abbreviations used in this paper: MHV, mouse hepatitis virus; CNS, central nervous system; CRG, cytokine-response gene; EAE, experimental autoimmune encephalomyelitis; GFAP, glial fibrillary acidic glycoprotein; IP-10, interferon-inducible protein-10; Mac, macrophage; MCP, macrophage-chemoattractant protein; MIP, macrophage-inflammatory protein; MS, multiple sclerosis; NDV, Newcastle disease virus; PFU, plaque-forming unit; p.i., postinfection; RPA, RNase protection assay.

  • Received July 10, 1997.
  • Accepted October 3, 1997.

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The Journal of Immunology
Vol. 160, Issue 2
15 Jan 1998
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