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The Journal of Immunology, 1999, 162: 1624-1632.
Copyright © 1999 by The American Association of Immunologists

The Inflammatory Response to Nonfatal Sindbis Virus Infection of the Nervous System Is More Severe in SJL Than in BALB/c Mice and Is Associated with Low Levels of IL-4 mRNA and High Levels of IL-10-Producing CD4+ T Cells1

Jennifer F. Rowell and Diane E. Griffin2

Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SJL mice are susceptible to inflammatory autoimmune diseases of the central nervous system (CNS), while BALB/c mice are relatively resistant. To understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of SJL and BALB/c mice to infection with Sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. Clearance of virus was similar, but SJL mice developed a more intense inflammatory response in the brain and spinal cord and inflammation persisted for several weeks. Analysis of lymphocytes isolated from brains early after infection showed an absence of NK cells in SJL mice, while both strains of mice showed CD4+ and CD8+ T cells. During the second week after infection, CD4+ T cells increased in SJL mice and the proportion of CD8+ T cells decreased, while the opposite pattern was seen in BALB/c mice. Expression of IL-10 mRNA was higher and IL-4 mRNA was lower in the brains of infected SJL than in BALB/c mice, while expression of the mRNAs of IL-6, IL-1ß, TNF{alpha}, and the Th1 cytokines IL-2, IL-12, and IFN-{gamma} was similar. Lymphocytes isolated from the CNS of SJL mice produced large amounts of IL-10. CNS lymphocytes from both strains of mice produced IFN-{gamma} in response to stimulation with Sindbis virus, but not in response to myelin basic protein. These data suggest that IL-10-producing CD4+ T cells are differentially recruited to or regulated within the CNS of SJL mice compared with BALB/c mice infected with Sindbis virus, a characteristic that may be related to low levels of IL-4, and is likely to be involved in susceptibility of SJL mice to CNS inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The AR339 strain of Sindbis virus (SV)3 is an alphavirus that causes acute, nonfatal, and usually asymptomatic encephalomyelitis in weanling mice. After intracerebral inoculation, the virus replicates rapidly in the brain and spinal cord, primarily in neurons. A systemic and local immune response is induced and virus replication is soon controlled with levels of infectious virus becoming undetectable within 7–8 days after infection. Elimination of infectious virus correlates with the appearance of neutralizing Ab and the development of a virus-specific mononuclear inflammatory response in the brain and spinal cord (1, 2). Adoptive transfer experiments using persistently infected immunodeficient SCID mice have shown that Ab specific for the E2 glycoprotein of SV mediates clearance of infectious virus, even in the absence of SV-specific cell-mediated immunity (1), so the role of the inflammatory response in recovery is not clear. Inflammation is first detectable 3–4 days after infection when mononuclear cells start to cross the cerebrovascular endothelium to form perivascular cuffs. The inflammatory response is maximal after 7–10 days and is usually resolved within 2–3 wk (2).

Inflammation in the central nervous system (CNS) is rigorously controlled by several known mechanisms and probably additional mechanisms yet to be identified. The blood brain barrier is a physical barrier that prevents entry of most immune cells. Activated T cells can pass through the blood brain barrier, but quickly exit the CNS in the absence of Ag recognition (3, 4). T cell stimulation by Ag present in the CNS is further inhibited due to low levels of MHC molecules, costimulatory molecules, and adhesion molecules, although cellular damage and inflammatory mediators can increase expression (5, 6).

SV infection results in varying levels of CNS inflammation depending on the strain of mouse infected. Compared with BALB/c mice, SJL mice display more extensive inflammation and this inflammation may persist for several weeks to months (7). SJL mice are also susceptible to several inflammatory autoimmune diseases of the CNS such as experimental autoimmune encephalitis (EAE) (8, 9) and Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination (10), while BALB/c and many other strains of mice are relatively resistant to these diseases. Both of these murine demyelinating diseases serve as models for the human demyelinating disease multiple sclerosis, and more complete understanding of the inflammatory process may enhance our general understanding of autoimmune diseases of the CNS.

EAE is induced by immunization with whole spinal cord or components of myelin such as myelin basic protein (MBP) or proteolipid protein together with CFA (8, 11). Paralysis is accompanied by inflammation consisting of T cells, macrophages and B cells. The disease is mediated by CD4+ Th1-type T cells that secrete inflammatory cytokines such as IL-2 and IFN-{gamma} (12). The inflammation persists for several days, then resolves.

TMEV infects neurons and oligodendroglia of the brain and spinal cord. Resistant strains of mice clear virus during the acute phase of the infection. In SJL mice, virus clearance is incomplete resulting in persistent infection of macrophages and glial cells. In these mice, TMEV-specific CD4+ Th1-type T cells mediate a chronic inflammatory process that results in demyelination and paralysis (13, 14).

The differences in the development and regulation of immune responses within the CNS of SJL mice compared with BALB/c mice that increase susceptibility to inflammatory diseases involve genes primarily outside the H-2 region and are only partially defined (10). To gain a better understanding of these differences, we have examined the CNS inflammatory response in SJL and BALB/c mice during acute encephalitis induced by infection with SV, a virus that is not associated with late neurologic disease. The immune response to SV results in clearance of virus in both strains of mice, but the inflammatory response to SV is more severe in SJL mice and SJL mice have lower levels of IL-4 mRNA, fewer NK cells, and more IL-10-producing CD4+ T lymphocytes in response to SV infection, suggesting altered immune regulation in the CNS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and viruses

SJL/J and BALB/cJ mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Four- to ten-week-old female mice were inoculated intracerebrally with 1000 plaque-forming units (PFU) of the AR339 strain of SV in 0.03 ml HBSS containing 1% FBS. Virus was grown and assayed using BHK-21 cells.

Histology and immunohistochemistry

At various times after infection, mice were anesthetized with methoxyflurane and perfused with PBS. Brains, spinal cords, and spleens were removed. All or part of each brain and spinal cord was either placed in 4% buffered paraformaldehyde for subsequent embedding in paraffin or rapidly frozen, together with the spleen, in OCT compound (Tissue-Tek; Miles, Elkhart, IN). Sections of paraffin-embedded tissue were stained with hematoxylin and eosin or with luxol fast blue, coded, and examined for inflammation and demyelination. Inflammation was scored on coded slides using a scale of 0–3 in which 0 indicates no detectable inflammation, 1 indicates one or two small inflammatory foci per section, 2 indicates moderate inflammatory foci in up to 50% of x10 fields, and 3 indicates moderate to large inflammatory foci in >50% of x10 fields. Scores were increased by up to 1 point with abundant parenchymal cellularity.

Cryopreserved sections were cut from OCT-embedded tissue and used for immunohistochemistry. Sections were blocked with 0.5% dry milk in PBS, and endogenous peroxidase activity was quenched with either 1% H2O2 in absolute methanol or 0.3% H2O2 in 0.1% sodium azide. Sections were incubated overnight at 4°C, with primary Ab diluted in PBS containing 0.5% dry milk, washed, and incubated with biotinylated secondary Ab (Vector Laboratories, Burlingame, CA) in PBS/0.5% dry milk. The signal was amplified using the Vectastain Elite ABC kit (Vector) and detected with 3,3'-diaminobenzidine (Sigma, St. Louis, MO). For detection of CD8, the signal was further amplified using TSA-Indirect Tyramide Signal Amplification (DuPont NEN, Boston, MA). The primary Abs used in this study are listed in Table IGo. Sections were examined under code for the level of inflammation, the proportion of positively staining cells in inflammatory lesions, and the relative number of positively staining cells in the parenchyma. Immunohistochemical staining was scored on coded slides using a scale of 0–3 in which 0 indicates no positive cells, 1 indicates 5–20%, 2 indicates 20–60%, and 3 indicates 60–100% positive inflammatory cells. Scores were adjusted by up to 1 point when the numbers of positively staining cells in the parenchyma were considerably higher or lower than average.


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  		Table I. Abs used for analysis of inflammatory cells and for ELISA

 
Isolation of lymphocytes from brain and spleen

Brain lymphocytes were isolated as previously described (15). Briefly, SV-infected mice were anesthetized with methoxyflurane and perfused with PBS. Brains were removed, homogenized through a mesh screen, and collected in HBSS containing 0.05% collagenase D (Boehringer Mannheim, Indianapolis, IN) and 10 µg/ml DNase I (Sigma). The brain homogenate was mixed at room temperature for 20 min, then allowed to settle for 20 min. The supernatant fluid was collected and layered onto a mixture of 75% Ficoll-Paque (Pharmacia, Piscataway, NJ) and 25% RPMI 1640 with 10% FBS. After centrifugation at 500 x g for 30 min, the overlying media and interface of tissue debris were removed, and cells in the remaining gradient media were washed with PBS/2% FBS. The pelleted cells were resuspended in PBS/2% FBS for flow cytometry or RPMI 1640/10% FBS for culture.

For isolation of spleen lymphocytes, spleens were removed from perfused mice, homogenized through a mesh screen, and collected in PBS. The homogenate was layered onto Lympholyte M density separation media (Cedarlane, Westbury, NY). After centrifugation, cells were collected from the interface, washed, and resuspended as described for brain lymphocytes.

Flow cytometry

For flow cytometry, 3–5 x 105 isolated lymphocytes in 100 µl PBS/2% FBS were incubated for 1 h at 4°C with FITC- or phycoerythrin (PE)-conjugated Abs as listed in Table IGo at 0.1 µg/ml. Conjugated isotype-matched control Abs at the same concentration were used to determine background staining. Stained cells were washed, resuspended in 0.5 ml PBS/2% FBS, and analyzed using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA). Debris was excluded and intact lymphocytes were included in the analysis using forward and side light scattering gates, and 5,000–10,000 gated events were collected for each sample. Percentages of positively staining cells were calculated by establishing quadrants on log-scale scatterplots of FITC vs PE fluorescence, which separated background staining from positive staining.

Cytokine mRNA expression

Brains from perfused mice were removed, frozen on dry ice, and subsequently homogenized in PBS. RNA was extracted from 1/4 of whole brain homogenate using RNA STAT-60 (Tel-Test "B", Friendswood, TX). cDNA was synthesized from 10% of total RNA as previously described (16) using avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim). Then, 5% of the cDNA was used for each PCR reaction that contained 200 µM of each dNTP, 1 µM of each specific primer, 2.5 U Taq polymerase (Boehringer Mannheim), and buffer as supplied by the manufacturer. PCR was performed using a 9600 thermal cycler (Perkin-Elmer, Cetus, Norwalk, CT). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA detection was used to control for varying amounts of input RNA. The optimal number of cycles needed for amplification in the linear range was determined for each cytokine and for GAPDH. Standards were included with each PCR reaction to ensure that amplification was occurring in the linear range. Primers and probes for GAPDH, IL-1ß, IL-2, IL-4, IL-6, IL-10, TNF{alpha}, and IFN-{gamma} and primers for the chemokines macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, and IFN-{gamma}-inducible protein-10 (IP-10) have been described previously (16, 17). Primers and probe for IL-12 were: sense, 5'-TCTGCAGAGAAGGTCACACTG; anti-sense, 5'-GACTTCGGTAGATGTTTCCTC; probe, 5'-CCTGATGAAGAAGCTGGTGCTGTAGTTCTC. For cytokines, PCR products were identified and quantitated relative to GAPDH as previously described (16) except that probes were labeled with digoxigenin and detected with alkaline phosphatase-labeled Ab to digoxigenin and the chemiluminescent substrate disodium 3-(4-methoxyspirodecan)phenyl phosphate (Boehringer Mannheim). For chemokines, PCR products were visualized on gels by staining with ethidium bromide.

Functional assays using brain lymphocytes

For determination of IL-10 secretion, lymphocytes isolated from brains or spleens of infected mice were added to 96-well plates at 5 x 105/ml in RPMI 1640 plus 10% FBS with or without 5 µg/ml ConA (Sigma) or 100 ng/ml phorbol myristic acid (Sigma) plus 2 µg/ml ionomycin (Sigma). After incubation for 3 days, supernatant fluids were collected and the amount of IL-10 was determined by ELISA using capture and detecting Abs shown in Table IGo following the protocol provided by the manufacturer. rIL-10 (PharMingen, San Diego, CA) was used to generate a standard curve.

For determination of Ag specificity, mononuclear cells were isolated from spleens of uninfected mice and T cells were depleted by treatment with rabbit anti-Thy1 antiserum (1:20; Cedarlane) at 4°C followed by incubation at 37°C with Low-Tox-M rabbit complement (1:10; Cedarlane). Lysed cells were removed using Lympholyte M as described above. Lymphocytes isolated from brains of infected mice (5 x 105/ml) were combined with autologous T cell-depleted spleen cells (2.5 x 106/ml) and incubated with or without UV-inactivated SV equivalent to 2 x 107 or 8 x 107 PFU/ml or with human MBP at 25 µg/ml. After incubation for 3 days, supernatant fluids were collected and IFN-{gamma} production was analyzed by ELISA (Endogen, Woburn, MA).

Detection of apoptosis

Brains and spinal cords removed from perfused mice were fixed in 4% paraformaldehyde and embedded in paraffin. Deparaffinized sections were subjected to terminal deoxynucleotidyltransferase-mediated UTP nicked end-labeling (TUNEL) as described previously (18). This technique detects endonucleolytically cleaved chromosomal DNA characteristically found in the nuclei of apoptotic cells. Staining was quantitated on coded slides by scoring the proportion of positive cells in inflammatory loci on a scale of 0–3 as for immunohistochemistry and the relative number of positive cells in the parenchyma on a scale of 0–3 in which 0 indicates no positive cells and 3 indicates the maximum number seen among all sections. These two scores were averaged for each section, then adjusted by up to 1 point if background staining was unusually high or low.

For annexin V staining, lymphocytes were isolated from brain and stained with PE-conjugated anti-CD3e for flow cytometry as described. Cells were then washed with PBS and incubated with FITC-conjugated annexin V diluted 1:10 in manufacturer-provided binding buffer from an Apoptosis Detection Kit (R & D Systems, Minneapolis, MN). Cells were diluted with binding buffer and analyzed immediately by flow cytometry as described.

Plaque assay

Brains were removed from perfused mice, frozen on dry ice, and stored at -80°C. Thawed brains were homogenized in cold PBS to make 10% (w/v) homogenates, and dilutions were prepared immediately in DMEM containing 1% FBS and plaqued on BHK-21 cells.

ELISA and plaque neutralization assay for serum Ab

Blood was collected from anesthetized mice, and serum was isolated using microtainer serum separation tubes (Becton Dickinson). For ELISA, enzyme immunoassay microtitration plates (Costar, Cambridge, MA) were coated with polyethylene glycol-precipitated SV at 3 µg/ml. Serial dilutions of serum ranging from 1:10 to 1:3000 were added to coated plates. Specific Ab was detected with horseradish peroxidase-conjugated anti-mouse Ig, IgG1, or IgG2a (see Table IGo) followed by O-phenylenediamine dihydrochloride (Sigma). Relative titers were calculated from dilutions in the linear range using a hyperimmune serum as a standard.

For neutralization assays, serial dilutions of serum were incubated with a known amount of SV for 30 min at 37°C. PFU/ml were measured on BHK-21 cells, and dilutions of serum needed for 50% plaque reduction were determined.

Statistical analysis

Student’s two-tailed test was used to assess significance of differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histopathology during SV infection

Inflammation in the brains and spinal cords of SV-infected SJL and BALB/c mice was assessed by routine histology and by immunohistochemistry at various times after infection. Total inflammation was quantitated using the number and size of inflammatory lesions in each tissue section (Fig. 1GoA). Inflammation was greater in SJL mice (day 5, p < 0.002; day 8, p = 0.02) than in BALB/c mice. Inflammatory lesions were first detected on day 3, and the level of inflammation reached a peak on day 5 in both strains. However, at the time of maximal inflammation, SJL mice had extensive infiltration of mononuclear cells typically seen during fatal infection with a neurovirulent strain of SV (Fig. 1GoB), while BALB/c mice showed only moderate inflammation typical of nonfatal infection (Fig. 1GoC) (16). In BALB/c mice, the inflammatory response was essentially resolved 2–3 wk postinfection (PI), but remained at moderate levels in SJL mice until 4–5 wk PI in most mice and for several weeks longer in a few mice. By 2 wk PI, inflammation in BALB/c mice consisted mostly of increased cellularity in the parenchyma. In contrast, SJL mice continued to have significant perivascular cuffing for the duration of the inflammatory response. No demyelination was observed in either strain at any time point.



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  		FIGURE 1. Inflammation in the CNS of SV-infected SJL and BALB/c mice. A, Quantitation of inflammation in hematoxylin and eosin-stained sections of brains and spinal cords from SJL and BALB/c mice. Inflammation was scored under code on a scale of 0–3: 0, no detectable inflammation; 1, one or two small inflammatory foci per section; 2, moderate-sized inflammatory foci in up to 50% of x10 fields; 3, moderate to large inflammatory foci in >50% of x10 fields. Scores were increased by up to 1 point for abundant parenchymal cellularity. Between 3 and 14 days PI, each point represents the average score for three to six mice from two separate experiments, and SEs are shown (day 5, p < 0.002; day 8, p = 0.02). After 14 days PI, each point represents the average score for two mice. B and C, Representative hematoxylin and eosin-stained sections of brains from an SJL (B) and BALB/c (C) mouse at day 6 PI (magnification, x400.

 
Immunohistochemistry was used to identify the immune cells present in inflammatory lesions in SJL and BALB/c mice, and the relative numbers of B cells, macrophages, CD4+ T cells, and CD8+ T cells were determined (Fig. 2Go). Cells expressing the B cell marker B220 were present at similar levels in SJL mice and BALB/c mice, and the levels remained moderate through 4 wk PI. Macrophages, identified by reactivity with the Ab F4/80, increased in number during the first week PI, and levels were similar in SJL and BALB/c mice. Cells expressing CD8 were also present at similar levels in both strains. In contrast, cells expressing CD4 were more abundant in SJL mice than in BALB/c mice. Many inflammatory lesions in brain and spinal cord sections from SJL mice consisted of 60–80% CD4+ T cells (Fig. 3GoA), while CD4+ T cells made up only 20–30% of cells in lesions from BALB/c mice (Fig. 3GoB). The levels of CD4+ T cells in BALB/c mice were similar to those seen in C57BL/6 mice and BALB/cBy mice (data not shown). The representation of CD4+ T cells was notably higher in SJL mice than in BALB/c mice at all time points with detectable inflammation and remained at near maximal levels from 7 to 25 days PI (Fig. 2Go).



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  		FIGURE 2. Identification of inflammatory cells by immunohistochemistry. Brain and spinal cord sections from infected SJL and BALB/c mice were stained for the immune cell markers indicated, coded, and representation of each type quantitated. Quantitation was on a scale of 0–3: 0, no positive cells; 1, 5–20% of inflammatory cells positive; 2, 20–60% positive; and 3, 60–100% positive. Scores were adjusted by up to 1 point when the numbers of positively staining cells in the parenchyma were substantially higher or lower than average. Through day 14 PI, each point represents the average score for three mice, and, after day 14, each point represents the average score for two mice. For CD4+ cells: day 5, p = 0.02; day 8, p = 0.1, day 14, p = 0.005.

 


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  		FIGURE 3. CD4+ T cells in brains of SJL and BALB/c mice at day 5 PI. Immunoperoxidase staining with Ab to L3T4(CD4) in SJL (A) and BALB/c (B) (magnification, x160).

 
Isolation of lymphocytes from brains of infected mice

Lymphocytes were isolated from the brains of SV-infected SJL and BALB/c mice by density gradient separation and characterized by flow cytometry. This allowed for a more quantitative characterization of the types of immune cells present in the brains of these mice. Isolated cells were double-stained for expression of CD3 together with CD4, CD8, B220, {alpha}ß or {gamma}{delta} TCR, or a pan NK cell marker. The proportion of isolated lymphocytes expressing each marker was determined at time points between 4 and 14 days PI (Fig. 4Go). Because debris from brain tissue remained in the cell preparations, only cells with light scattering characteristics of lymphocytes were analyzed. At most time points, 70–80% of the cells were identifiable. However, on day 4 PI only 50% of the cells were identifiable due either to the presence of a larger proportion of unidentified cells or to the presence of proportionately more debris within the light scattering gate because fewer lymphocytes were isolated on day 4 PI.



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  		FIGURE 4. Identification by flow cytometry of lymphocytes isolated from brains of SV-infected SJL and BALB/c mice. A, Isolated lymphocytes were labeled with PE-conjugated anti-CD3e and FITC-conjugated anti-NK. The percentages of events staining positively with each Ab are shown. B, Isolated lymphocytes were labeled with PE-conjugated anti-CD3e and either FITC-labeled anti-CD4 or FITC-labeled anti-CD8. The percentages of CD3+ events staining positively for CD4 or CD8 are shown. Each point represents the average of results from two to four mice. For CD4+ T cells: day 10, p = 0.001; day 14, p = 0.005. For CD8+ T cells: day 10, p = 0.001; day 14, p = 0.005.

 
The percentage of CD3+ cells increased during the first week PI and remained elevated through the second week. At early time points, the percentage of lymphocytes that were CD3+ was higher in SJL mice than in BALB/c mice (day 7, p = 0.001) (Fig. 4GoA). By day 10, the proportion of CD3+ cells was similar in both strains. Parallel to this observation, the proportion of lymphocytes that were NK cells was higher in BALB/c mice (day 6, p = 0.002) (Fig. 4GoA), consistent with earlier studies showing that SJL mice have low, poorly inducible NK activity (19). In contrast to what was seen with immunohistochemistry, the percentage of B220-expressing cells was very low in both strains at all times examined (data not shown). Staining for IgM resulted in similarly low levels of positive cells, indicating that B cells may not be isolated efficiently by the technique used.

The composition of the CD3+ cells was further studied by staining for CD4 or CD8 (Fig. 4GoB). The proportion of CD4+ and CD8+ cells was similar in the two mouse strains through day 7 PI. After day 7, the proportion of CD4+ cells increased in SJL mice, while the proportion of CD8+ cells increased in BALB/c mice. A late increase in the proportion of CD8+ T cells in the brains of BALB/c mice was also seen in earlier flow cytometry, but not in immunocytochemical, studies (15, 20). Staining of CD3+ cells for {alpha}ß TCR and {gamma}{delta} TCR showed that in both strains of mice the majority of CD3+ cells expressed {alpha}ß TCR with <10% expressing {gamma}{delta} TCR (data not shown).

Cytokine mRNA expression by resident and inflammatory cells in the CNS

To characterize further the CD4+ T cells present at increased levels in SJL mice and to determine whether early responses of resident CNS to infection were different, cytokine mRNA expression was analyzed by semiquantitative RT-PCR of RNA isolated from brains of infected mice (Fig. 5Go). Early expression of IL-1ß, IL-6, IL-12, and TNF{alpha} mRNAs, proinflammatory cytokines likely to be important for recruitment of inflammatory cells, was identical. Expression of the mRNAs of three chemokines, believed to be important in the recruitment of T cells and the development of EAE, MIP-1{alpha}, MIP-1ß, and IP-10 (21, 22), was measured, and no differences were seen (data not shown). At days 5 and 8 PI, after initiation of the inflammatory responses, expression of IL-10 was higher in SJL mice (day 5, p = 0.002). Expression of IL-4 was consistently higher in BALB/c mice (day 1, p = 0.001; day 3, p = 0.01). Expression of IL-12, IFN-{gamma}, and TNF{alpha} mRNAs increased to similar levels in both strains.



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  		FIGURE 5. Expression of cytokines in brains of SV-infected SJL and BALB/c mice. The relative levels of cytokine mRNAs as determined by semiquantitative RT-PCR. Each point represents the average for three mice.

 
Because the presence of cytokine mRNA may not reflect synthesis of protein, IL-10 expression was examined further by measuring IL-10 production by lymphocytes isolated from brains of infected mice at day 10 PI and stimulated with phorbol ester and ionomycin (Fig. 6Go). Secretion of IL-10 by lymphocytes from BALB/c brains was undetectable, while secretion by lymphocytes from SJL brains was high and similar to secretion by lymphocytes isolated from spleens of both strains of mice.



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  		FIGURE 6. Production of IL-10 by brain lymphocytes. Lymphocytes isolated from brains at day 10 PI were stimulated in vitro with PMA and ionomycin, and IL-10 was measured in supernatant fluids collected at 72 h. Each bar represents cells from one spleen or four brains, each divided into three wells (for brain p = 0.001).

 
Ag specificity of brain lymphocytes

Earlier studies had suggested that autoimmune T cells recognizing MBP were activated in SJL mice during SV infection (7). To determine whether the increased numbers of CD4+ T cells seen in the CNS of SJL mice were virus-specific or recognized the autoantigen MBP, we tested the ability of CNS lymphocytes to recognize SV proteins and MBP (Fig. 7Go). Lymphocytes isolated from the brains of mice 10 days after infection were cultured with autologous T cell-depleted spleen cells in the presence or absence of UV-inactivated SV or MBP to stimulate T cells through the MHC class II pathway. Ag recognition was measured by secretion of IFN-{gamma}. Both SJL and BALB/c lymphocytes secreted IFN-{gamma} in response to SV in a dose-dependent manner, and neither responded to MBP. No IFN-{gamma} secretion was detected in the absence of Ag. SJL lymphocytes secreted higher levels of IFN-{gamma} in response to SV than BALB/c lymphocytes, approximately proportionate to the higher levels of CD4+ T cells in the SJL brain lymphocyte population.



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  		FIGURE 7. Ag specificity of CD4+ T cells isolated from brains of SV-infected mice. Lymphocytes isolated at 10 days PI were incubated with T cell-depleted autologous spleen cells alone or with 108 PFU (1x) or 4 x 108 PFU (4x) UV-inactivated SV or 25 µg MBP. After 3 days, supernatant fluids were analyzed for the presence of IFN-{gamma} by ELISA. Each bar represents cells from two or three brains combined and divided into three wells.

 
Apoptosis of inflammatory cells

T cells and macrophages present in CNS inflammation during EAE undergo apoptosis during the recovery phase of the disease (23, 24). To determine whether the increased inflammation seen in SV-infected SJL mice was due to reduced death of infiltrating inflammatory cells, apoptotic cells were quantitated by two methods during the second week PI. First, brain and spinal cord sections from SV-infected SJL and BALB/c mice were stained using the TUNEL technique (Fig. 8GoA). In tissue sections from both strains, a small proportion of cells in inflammatory lesions stained positively. In addition, TUNEL-positive cells with lymphocyte morphology were scattered throughout the parenchyma. TUNEL staining was scored on coded sections by identifying the proportion of positive cells in inflammatory foci together with the relative numbers of positive cells in the parenchyma and was similar during this period of time in the two mouse strains.



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  		FIGURE 8. Apoptosis of inflammatory cells in the CNS of SV-infected mice. A, TUNEL staining of inflammatory cells in brain and spinal cord sections at various times PI. Apoptotic cells were identified by TUNEL staining and quantitated as described in Materials and Methods. Each point represents the average score for three mice. B, Annexin V staining of lymphocytes isolated from brains of infected mice. Lymphocytes were stained with FITC-conjugated annexin V and PE-conjugated anti-CD3e. The percentages of CD3+ cells that stained with annexin are shown. Each point represents the average of results from two mice.

 
Preapoptotic T cells were identified by annexin V staining of lymphocytes isolated from the brains of infected mice (Fig. 8GoB). Annexin V binds to phosphotidylserine expressed on the cell surface during the early stages of apoptosis, and the level of binding can be measured using flow cytometry (25). A similar small proportion of CD3+ cells from the brains of both mouse strains stained with annexin V.

Virus replication and clearance

Differences in virus replication or clearance could change the degree or persistence of the inflammatory response. To assess this possibility, infectious virus in brain homogenates was measured by plaque assay at various times after infection (Fig. 9GoA). The levels of infectious virus and the time course of viral clearance were identical between the two strains of mice.



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  		FIGURE 9. Virus clearance and Ab response to virus. A, Levels of infectious virus in brain homogenates at various times PI. Each point represents the average of results from three mice. Infectious virus was undetectable at day 8 PI. B, SV-specific serum Ab titers as detected by ELISA. Titers were determined relative to a standard hyperimmune serum. Each point through day 14 PI represents the average titer for three mice, and points after day 14 PI represent the average titer for two mice. Ab was undetectable in BALB/c mice and barely detectable in SJL mice at day 3 PI.

 
Although antiviral Ab has been shown to be sufficient for clearance of SV in SCID mice (1), virus-specific T lymphocytes might compensate for an inadequate Ab response and contribute to virus clearance. To determine whether the Ab response to SV differs between SJL and BALB/c mice, levels of SV-specific Ab in the sera of infected mice were measured by ELISA (Fig. 9GoB). In both strains, Ab levels increased during the first 1–2 wk PI and remained high thereafter. There were no differences in Ab titers at any time point. The levels of SV-specific IgG1 and IgG2a were also determined by ELISA, and in both strains the majority of Ab was of the IgG2a isotype (data not shown). Levels of neutralizing Ab in the serum were determined by plaque neutralization, and again, no differences were seen (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
SJL mice are susceptible to several inflammatory diseases of the CNS, while other strains of mice such as BALB/c are relatively resistant (8, 9, 10). Susceptibility to induction of these autoimmune CNS diseases may be a consequence of inadequate regulation of immune responses and inflammatory processes in the CNS. We sought to better understand this regulation by studying the response to SV, a well-characterized viral infection of the CNS that results almost invariably in asymptomatic encephalitis. SV infection of SJL mice resulted in more extensive and longer lasting mononuclear cell inflammation than occurred in BALB/c mice, even though there were no differences in virus growth, virus clearance, or Ab production. However, the characteristics of the cellular infiltration and cytokines produced were distinct. In SJL mice, the initial inflammatory response included very few NK cells and, over time, CD4+ T cells continued to accumulate in perivascular regions, and inflammation was prolonged. In BALB/c mice, NK cells were abundant early, CD4+ T cells were the predominant T cell early but were in the minority by 10 days PI, and inflammation was essentially resolved within 4 wk. BALB/c mice had consistently higher levels of IL-4 mRNA in the CNS than SJL mice. T cells isolated from the CNS of both strains of mice produced comparable amounts of IFN-{gamma} in response to Ag stimulation in vitro, but SJL CNS T cells also produced large amounts of IL-10. These studies demonstrate that CNS immune responses in SJL mice have inherent differences from those in BALB/c mice and provide insight into the pathogenesis of the inflammatory diseases to which SJL mice are susceptible.

CD4+ T cells were much more prevalent and persistent in the brains and spinal cords of SJL mice in response to SV infection. That this is a characteristic of SJL mice is suggested by the fact that prolonged CNS inflammation, without evidence of persistent infection, has also been reported in SJL mice infected with Semliki Forest virus, an alphavirus related to SV (26). The phenotypes of the cells responding to Semliki Forest virus infection were not characterized, but studies of inflammatory cells in the brains of TMEV-infected SJL mice have also shown fewer CD8+ and more activated perivascular CD4+ T cells than in other strains of mice (14, 27). Furthermore, CNS mononuclear inflammatory cells in SJL mice infected with SV are more likely to show evidence of proliferation than in BALB/c mice (28). Together, these data suggest that alterations in recruitment of CD4+ T cells into the CNS and regulation of their numbers after entry may contribute significantly to the generation of inflammatory disease in the brain and spinal cord.

The initial phase of inflammation induced by SV infection followed a similar time course. However, a considerable proportion of the lymphocytes found in BALB/c brains early in the inflammatory response were NK cells, while NK cells were nearly undetectable in SJL brains. The response to TMEV in SJL mice is also deficient in NK cell activity, and this is postulated to contribute to the defect in early virus clearance (29). Although NK cells and NK activity are prevalent in the CNS of BALB/c mice early after SV infection, there is no evidence that they are necessary for, or contribute to, SV clearance from the CNS (30, 31). NK cells secrete IFN-{gamma} and TNF{alpha}, cytokines that may play a role in the initiation and control of T cell responses (32). However, analysis of cytokine mRNAs in the brain early after infection revealed no differences in IFN-{gamma} or TNF{alpha}. It is possible that the absence of NK cells in SJL mice may be involved in shifting the immune response to one dominated by CD4+ T cells.

We also found no differences in mRNA expression for IL-1ß or IL-6 or the chemokines MIP-1{alpha}, MIP-1ß, or IP-10. The only difference was lower IL-4 mRNA levels in SJL mice. This difference has also been observed in TMEV-infected mice (33) and may be due to the known SJL deficiency in CD4+ T cells expressing NK1.1, a subset that produces IL-4 efficiently early after TCR cross-linking (34, 35). Numerous studies have demonstrated the ability of IL-4 to promote the development of Th2 responses while inhibiting the development of Th1 responses (36). IL-4 knockout mice have an increased susceptibility to EAE with more intense CNS inflammation, suggesting that IL-4 also down-modulates inflammatory processes in the CNS (37). Therefore, low levels of IL-4 in the CNS may play an important role in the predisposition of SJL mice to increased and prolonged inflammation.

In EAE and TMEV-induced demyelination, disease-mediating T cells are of the Th1 subtype, secreting inflammatory cytokines such as IL-2 and IFN-{gamma} (12, 13). A role for antiviral CD4+ Th1 responses has also been suggested by an increased proportion of TMEV-specific Abs of the IgG2a rather than the IgG1 subclass (38). We measured cytokine expression to determine whether the CD4+ T cells present in the CNS of SJL mice during SV infection were also of the Th1 subtype. A comparison of the overall levels of CNS cytokine mRNA expression during the peak of T cell infiltration showed that expression of IL-2 and IFN-{gamma} mRNAs, cytokines typically expressed by Th1 cells, was not elevated in SJL mice. However, expression of Th2 cytokines IL-4 and IL-10 was different. IL-10 mRNA and protein were increased while IL-4 mRNA was decreased. The study of CNS cytokine mRNAs in response to TMEV infection has also shown lower levels of IL-4 mRNA and higher levels of IL-10 mRNA during TMEV-induced disease in SJL mice compared with BALB/c mice (33).

The increased expression of IL-10 mRNA in SV-induced encephalitis was due to T cells because large amounts of IL-10 were produced in culture by lymphocytes isolated from brains of infected SJL, but not BALB/c, mice. Isolated lymphocytes also secrete IFN-{gamma} in response to stimulation with SV in culture, and both strains of mice produced antiviral Ab primarily of the IgG2a subclass. Thus, the CD4+ T cells present at increased levels in the CNS of SJL mice during SV infection do not appear to be predominantly Th1 or Th2 cells. Although we do not know whether IFN-{gamma} and IL-10 are produced by the same population of T cells in SJL mice, CD4+ T cells secreting this combination of cytokines have been reported by murine clones suppressing autoimmune diabetes (39) and by human lines and clones responding to chronic infection or primed by IL-12 (40, 41, 42).

The effect of IL-10 on inflammatory processes is complex. Although generally regarded as a Th2 cytokine that down-regulates Ag presentation and production of proinflammatory cytokines by macrophages (43), IL-10 also up-regulates macrophage production of nitric oxide and expression of Fc{gamma}R (44, 45). IL-10 inhibits the ability of microglial cells to express MHC class II, to produce IL-12, to present Ag to T cells and to activate astrocytes (46, 47, 48). IL-10 down-regulates T cell responses in vivo. When given systemically IL-10 synergizes with IL-4 to inhibit delayed-type hypersensitivity responses (49, 50). IL-10 also stimulates B cell proliferation and induces Ig secretion (51). Because the presence of Ab is critical for control of SV replication (1), inducing Ab production through IL-10 secretion may be a major role for CD4+ T cells in the CNS of infected mice.

During monophasic inflammatory processes, IL-10 tends to be expressed at the highest levels late in inflammation. An increase in IL-10 expression in the CNS is observed during and after recovery from EAE (52). Apoptosis of activated cells is believed to be essential for down-regulation of most immune responses and appears to play an important role in regulation of inflammation in immune privileged sites (53). Apoptosis of T cells in inflammatory lesions of EAE along with increased production of IL-10 has been associated with decreasing inflammation and recovery from disease (23, 24, 52). IL-10 promotes activation-induced death of T cells and can prevent or treat autoimmune diseases, including EAE (54, 55, 56). Expression of IL-10 by T cells in SJL brains may, therefore, be a response to the high levels of inflammation and represent an attempt to down-regulate that process.

We found no evidence that SJL lymphocytes in the CNS exhibited increased apoptosis during the recovery phase, suggesting that the increased IL-10 produced did not effectively control the inflammatory process. Thus, the enhanced inflammation seen in SJL mice is most likely to result from increased entry of activated SV-specific IL-10-producing CD4+ T cells into the CNS of SJL mice from the periphery and perhaps local expansion of this population after entry. This increase in CNS inflammation is correlated with low levels of IL-4.


   Footnotes
 
1 This work was supported by Research Grant NS29234, a Javits Neuroscience Investigator Award from the National Institutes of Health (D.E.G.), and by a postdoctoral fellowship from the National Multiple Sclerosis Society (J.F.R.). Back

2 Address correspondence and reprint requests to Dr. Diane E. Griffin, Department of Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205. E-mail address: Back

3 Abbreviations used in this paper: SV, Sindbis virus; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; GAPDH, glyceraldehyde phosphate dehydrogenase; IP, IFN-{gamma}-inducible protein; MBP, myelin basic protein; MIP, macrophage inflammatory protein; PE, phycoerythrin; PFU, plaque-forming units; PI, postinfection; TMEV, Theiler’s murine encephalomyelitis virus; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP nicked end labeling. Back

Received for publication June 17, 1998. Accepted for publication October 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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