Expression of Mig (Monokine Induced by Interferon-γ) Is Important in T Lymphocyte Recruitment and Host Defense Following Viral Infection of the Central Nervous System

Michael T. Liu, David Armstrong, Thomas A. Hamilton and Thomas E. Lane
J Immunol February 1, 2001, 166 (3) 1790-1795; DOI: https://doi.org/10.4049/jimmunol.166.3.1790

Abstract

Induction of a Th1 immune response against viral infection of the CNS is important in contributing to viral clearance. The present studies demonstrate a role for the T cell chemoattractant chemokine Mig (monokine induced by IFN-γ) in contributing to a Th1 response against mouse hepatitis virus infection of the CNS. Analysis of the kinetics of Mig expression revealed mRNA transcripts present at days 7 and 12 postinfection (p.i.) but not early (day 2) or late (day 35) in the infection. To determine functional significance, mouse hepatitis virus-infected mice were treated with anti-Mig antisera, and the severity of disease was evaluated. Such treatment resulted in a marked increase in mortality that correlated with a >3 log increase in viral burden within the brains as compared with control mice treated with normal rabbit serum. Anti-Mig-treated mice displayed a significant decrease (p < 0.005) in CD4+ and CD8+ T cell recruitment into the CNS as compared with normal rabbit serum-treated mice. In addition, anti-Mig treatment resulted in a significant decrease (p < 0.05) in levels of IFN-γ and IFN-β that coincided with increased (p < 0.02) expression of the anti-inflammatory Th2 cytokine IL-10 within the CNS. Collectively, these data indicate that Mig is important in contributing to host defense by promoting a protective Th1 response against viral infection of the CNS.

Leukocyte infiltration into the CNS in response to viral infection is an important component of host defense and is often required for elimination of virus. However, the molecular mechanisms governing the development of inflammatory events following viral infection of the CNS are poorly understood. Infection of the CNS with mouse hepatitis virus (MHV),3 a (+) strand RNA virus, induces a robust cell-mediated response that is essential in control of viral growth. Both CD4+ and CD8+ T lymphocytes are required for optimal clearance of virus from the CNS, and one mechanism by which T lymphocytes participate in the elimination of MHV is through the release of IFN-γ (1, 2, 3, 4, 5, 6). Therefore, the collective evidence indicates that a Th1-mediated response against MHV infection of the CNS is critical in host defense and viral clearance.

Chemokines represent an ever-growing family of chemotactic cytokines that have been shown to target specific populations of leukocytes during periods of inflammation (7, 8). A recent study has demonstrated that an orchestrated expression of chemokine genes occurs following MHV infection of the CNS, suggesting that these molecules function to attract leukocytes into the CNS in response to viral infection (9). In support of this is a recent study demonstrating that expression of the non-ELR (glutamic acid-leucine-arginine) CXC chemokine IP-10 (IFN-γ-inducible protein 10 kDa) is important in initiating a Th1 response characterized by T lymphocyte infiltration and production of the IFN-γ in response to MHV infection of the CNS (4). Mig (monokine induced by IFN-γ) is another non-ELR CXC chemokine that shares similarities with IP-10 (10). However, although IP-10 expression is inducible by both IFN-αβ and IFN-γ, Mig expression in strictly dependent on IFN-γ (10, 11, 12, 13, 14). Both Mig and IP-10 exert a potent chemotactic effect on T lymphocytes by binding to the shared receptor CXC chemokine receptor 3 (CXCR3) (10, 15, 16, 17). Expression of IP-10 and Mig is thought to contribute to a variety of inflammatory pathologies by attracting leukocytes to sites of infection or injury (18, 19, 20, 21, 22). Moreover, both IP-10 and Mig have been shown to exhibit antitumor properties as well as being important contributors to antiviral defense (4, 23, 24, 25, 26).

Given the similarities as well as the differences between IP-10 and Mig with regard to biochemical and functional properties, studies were performed to investigate the contributions of Mig in host defense following MHV infection of the CNS. The results presented indicate that, unlike IP-10, which is expressed during acute and chronic disease, Mig expression is limited to the acute stage of disease. Neutralization of Mig activity through administration of rabbit-specific antisera resulted in a marked increase in mortality and delayed viral clearance that correlated with a significant decrease in T lymphocyte infiltration into the brain. In addition, anti-Mig treatment resulted in a significant decrease in mRNA transcripts and protein for IFN-γ and IFN-β that coincided with a significant increase in the anti-inflammatory Th2 cytokine IL-10. Together, these results indicate Mig is an important component in CNS host defense following viral infection of the CNS by contributing to the development of a protective Th1 response.

Materials and Methods

Virus and mice

The MHV strain V5A13.1 (referred to henceforth as MHV) was provided by M. Buchmeier (Scripps Research Institute, La Jolla, CA) (27). Age-matched (5–7 wk), male C57BL/6 mice (H-2b background) used for the studies described were purchased from Sprague-Dawley (San Diego, CA). Following anesthetization by inhalation of methoxyflurane (Pitman-Moore, Washington Crossing, NJ), mice were injected intracranially with 10 PFU of MHV suspended in 30 μl of sterile saline (9). Control (sham) animals were injected with sterile saline alone. Animals were sacrificed at days 3, 7, 9, and 10 postinfection (p.i.), at which point brains and spinal cords were removed. One-half of each brain was used for plaque assay on the DBT astrocytoma cell line to determine viral burden (1, 28). The remaining halves were either fixed for histologic analysis, stored at −80°C for RNA isolation, or used for FACS analysis.

Ab preparation and treatment of mice

Rabbit antisera to Mig were produced by Biosynthesis (Lewisville, TX) using a synthetic peptide (CISTSRGTIHYKSLK, coupled to carrier protein keyhole limpet hemocyanin) selected from the Mig protein sequence (24). This reagent has previously been shown to be specific for Mig and does not cross-react with IP-10, RANTES, or other known chemokines (24). MHV-infected mice were divided into two groups and treated with either normal rabbit serum (NRS) or anti-Mig. Mice were injected i.p. with 0.5 ml of Ab (0.5 mg/ml) on days 0, 2, 5, 7, and 9 p.i.

Combined in situ hybridization and immunohistochemistry

For studies designed to colocalize cellular Ags with in situ signal for Mig mRNA transcripts, immunohistochemical analysis preceded in situ hybridization using a previously described protocol (9). Staining for glial fibrillary acidic protein (GFAP) (rabbit polyclonal anti-bovine GFAP; Dakopatts, Carpinteria, CA) was performed on brain sections fixed in 10% normal buffered formalin and embedded in paraffin (9). PBS used for dilution of anti-GFAP (1:1000) as well as in washing steps was diethyl pyrocarbonate treated to reduce RNase contamination and loss of in situ signal. Following application of the chromagen diaminobenzidine, slides were washed twice in PBS and then prehybridized 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 (9). Upon development, the slides were counterstained in hematoxylin only, dehydrated, and mounted.

T cell isolation and flow cytometry

Cells were obtained from brains of mice treated with either anti-Mig or NRS at days 3, 7, and 9 p.i. A single-cell suspension was obtained by a previously described method (1), and FITC-conjugated rat anti-mouse CD4 and CD8 were used to detect infiltrating CD4+ and CD8+ T cells. As a control, an isotype-matched FITC-conjugated Ab was used. Cells were incubated with Abs for 30 min at 4°C, washed, fixed in 1% paraformaldehyde, and analyzed on a FACStar (Becton Dickinson, Mountain View, CA) (1, 4).

Immunohistochemistry

Primary Abs (diluted in PBS containing 2% normal goat serum) used for immunohistochemical detection of cellular Ags were as follows: rat anti-mouse CD4 (PharMingen, San Diego, CA) at 1:200 and rat anti-mouse CD8a (PharMingen) at 1:100. Staining for CD4 and CD8 was performed on 8-μm frozen brain sections fixed in 95% ethanol for 10 min at −20°C. These reagents are capable of recognizing either mouse CD4 or CD8 (manufacturer’s specifications). A biotinylated secondary Ab was used (1:300; Vector Laboratories, Burlingame, CA) and the ABC Elite (Vector Laboratories) staining system was used according to manufacturer’s instructions. Diaminobenzidine was used as a chromagen. All slides were counterstained with hematoxylin, dehydrated, and mounted. Staining controls were omission of primary Abs from the staining sequence (original magnification ×40).

RT-PCR

Total RNA was extracted using Trizol reagent (Life Technologies, Grand Island, NY) and reverse transcribed using the avian myeloblastoma virus reverse transcriptase system (Promega, Madison, WI). PCR amplification was performed on resulting cDNA for 30 cycles with specific primers for either CXCR3(forward, 5′-GCGGCCGCAACTCTTCCATTGTGG; reverse, 5′-GAATTCAAGGCCCCTGCATAGAAGTT), L32 (forward, 5′-AACGCTCAGCTCCTTGACAT; reverse, 5′-AACCCAGAGGCATTGACAAC), or Mig (forward, 5′-CGT CGT CGT TCA AGG AAG; reverse, 5′-TCG AAA GCT TGG GAG GTT). Sequence analysis of CXCR3, L32, and Mig amplicons confirmed primer specificity. Amplification was performed on an automated Perkin-Elmer/Cetus (Norwalk, CT) model 480 DNA thermocycler using 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 1–3 were repeated 29 times for a total of 30 cycles and were followed by a 7-min incubation at 72°C. The Mig fragment was cloned into the pCR Script SK+ vector (Stratagene, San Diego, CA) and used for RNase protection assay (RPA) analysis as described.

RNase protection assay

Total RNA was extracted from brains and spinal cords of MHV-infected animals treated with either anti-Mig or NRS at days 2, 7, 12, and 35 p.i. (9). The antisense riboprobe used to detect Mig mRNA was derived by RT-PCR amplification of cDNA generated from total RNA isolated from the brain of an MHV-infected mouse at day 7 p.i. For analysis of Mig and MHV gene expression, the Mig riboprobe was used in combination with a previously described riboprobe specific for the MHV spike gene and IP-10 (5, 9). L32 was added as an internal control to verify consistency in RNA loading and assay performance (1, 9). Cytokine transcripts were analyzed using the mCK-3 multitemplate probe sets (PharMingen). RPA analysis was performed with 15 μg of total RNA using a previously described protocol (1, 9). For quantification of signal intensity, autoradiographs were scanned and individual chemokine bands were normalized as the ratio of band intensity to the L32 control (1, 4, 9). Analysis was performed using NIH Image 1.61 software.

ELISA

IFN-γ, IL-4, and IL-10 protein levels within experimental groups were determined using the Quantikine M mouse immunoassay kits (R&D Systems, Minneapolis, MN) (1, 4). Tissue samples were homogenized in 1 ml of sterile PBS and spun at 400 × g for 5 min at 4°C. Duplicate supernatant samples were used to determine respective protein levels present within the tissues according to the manufacturer’s instructions. Following the enzymatic color reaction, samples were read at 450 nm and respective protein levels were quantified in comparison to a standard curve (supplied by the manufacturer). The limit of sensitivity of protein detection was ∼8.0 pg/ml. The reagents used for these experiments do not cross-react with other mouse cytokines (manufacturer’s specifications).

Results

MHV infection and Mig expression

Following MHV infection of the CNS, viral titers peaked at day 7 p.i., yet virus could not be isolated by 10 days p.i. as determined by plaque assay (limit of detection ∼100 PFU/g tissue), which is similar with the kinetics of viral clearance determined in previous studies (Table I⇓) (4). During the acute stage of disease (≤day 7), virus replicates within neurons as well as glial cells (29, 30). Animals that survive acute disease often develop a chronic demyelinating disease characterized by viral persistence within astrocytes and oligodendrocytes accompanied by mononuclear cell infiltration and myelin destruction (29, 31). Total RNA was isolated from brains at days 2, 7, 12, and 35 days p.i., and the kinetics of Mig, IP-10, and viral gene expression was determined by RPA. The results presented in Fig. 1⇓ demonstrate that MHV spike gene and IP-10 expression is detected as early as day 2 p.i., yet Mig is not detected at this time point. However, Mig, IP-10 and the MHV spike gene are prominently expressed at days 7 and 12 p.i. By day 35 p.i., a time in which the animals exhibit extensive myelin loss (data not shown), Mig transcripts are undetectable, whereas spike gene and IP-10 transcripts are still present. Double-labeling using an antisense riboprobe specific for Mig mRNA and a polyclonal Ab to GFAP indicate that astrocytes express Mig following MHV infection (Fig. 2⇓). Greater than 95% of cells expressing Mig transcripts were found to be astrocytes as determined by counting double-positive cells within the brains of infected mice. Although double-labeling was not performed, it is likely that macrophages are the additional cellular sources of Mig expression within the CNS of infected mice (10). Collectively, these data suggest that Mig expression is important during acute disease but does not contribute to chronic demyelination within persistently infected mice.

FIGURE 1.
FIGURE 1.

Mig and IP-10 expression. Densitometric analysis of RPA autoradiograph showing MHV spike, IP-10, and Mig transcripts present in the brains of sham and MHV-infected mice. Data is presented as normalized units representing the ratio of signal intensity of MHV spike, IP-10, or Mig to internal L32 included in the probe set. Values were obtained from the scanned autoradiograph using NIH Image 1.61 software (1 ,9 ). Data are presented as mean ± SEM. (Sham, n = 2; PID 2, n = 2; PID 7, n = 3; PID 12, n = 2; PID 35, n = 3).

FIGURE 2.
FIGURE 2.

Astrocytes express Mig. Representative section from the brain of a MHV-infected mouse at 7 days p.i. showing astrocyte expression of Mig in response to infection. Anti-GFAP staining was performed on tissue fixed in 10% normal buttered formalin followed by the addition of [35S]UTP-labeled antisense riboprobe specific for Mig. Brown cells (arrowheads) are indicative of astrocytes. Overlaying silver grains (large arrow) represent astrocyte expression of Mig. Original magnification ×40.

View this table:
Table I.

Delayed viral clearance in anti-Mig-treated mice

Anti-Mig treatment and neurologic disease

MHV-infected mice were treated with anti-Mig antisera in an attempt to evaluate the functional significance of Mig expression following viral infection of the CNS. Such treatment resulted in a marked increase in mortality as compared with control mice treated with NRS (Fig. 3⇓). By day 12 p.i., <10% of anti-Mig-treated mice survived the infection in contrast to an approximate 50% survival rate in infected mice treated with NRS (Fig. 3⇓). Surviving mice treated with anti-Mig displayed significantly higher (p < 0.001) titers of virus (5.4 ± 0.4, n = 4) as compared with titers present in NRS-treated mice (2.0 ± 0.2, n = 10) (Table I⇑). These data indicate that neutralization of Mig during acute disease results in increased mortality that correlates with an increase in viral burden within the CNS.

FIGURE 3.
FIGURE 3.

Increased mortality in anti-Mig-treated mice. Mice were infected intracranially with 10 PFU of MHV and treated i.p. with either anti-Mig or NRS at days 0, 2, 5, 7, and 9 p.i. By 12 days p.i., ∼50% of NRS-treated mice survived the infection, whereas <10% of anti-Mig-treated mice survived (Anti-Mig, n = 27; NRS, n = 21).

Anti-Mig treatment results in decreased T lymphocyte infiltration into the CNS

We have previously shown that CD4+ and CD8+ T lymphocytes present within the CNS of MHV-infected mice express CXCR3, the shared receptor for Mig and IP-10 (4). These data suggest that Mig is able to attract T cells into the CNS in response to viral infection. To determine T lymphocyte infiltration into the CNS, FACS analysis was performed at 3, 7, and 9 days p.i. At 3 days p.i. no appreciable difference in T lymphocyte infiltration was observed between anti-Mig- and NRS-treated mice (Table II⇓). This observation is not unexpected as Mig expression is not detected at this point (Fig. 1⇑). In contrast, at 7 and 9 days p.i., MHV-infected mice treated with anti-Mig displayed significantly lower levels of both CD4+ (45 and 53% decrease at 7 and 9 days p.i., respectively) and CD8+ (60 and 40% decrease at 7 and 9 days p.i., respectively) T lymphocyte infiltration into the CNS when compared with levels present within NRS-treated mice (Table II⇓). This decrease in infiltration was further confirmed using immunohistochemical staining for either CD4 or CD8 Ag within brains of mice treated with either anti-Mig or NRS (Fig. 4⇓). Consistent with the FACS data, increased numbers of CD4+ and CD8+ T lymphocytes were detected within perivascular cuffs present within the brains of NRS-treated mice as compared with anti-Mig-treated mice. T lymphocytes that are present within the CNS of anti-Mig-treated mice migrate to similar anatomic areas of the brain as control mice, e.g., areas where viral Ag is detected, indicating tissue distribution of infiltrating T cells is not affected (data not shown).

FIGURE 4.
FIGURE 4.

Decreased levels of infiltrating T lymphocytes within the CNS of anti-Mig-treated mice. Shown are representative sections of a perivascular cuff in the brains of MHV-infected mice treated with either anti-Mig or NRS at 7 days p.i. Top, Brain from NRS-treated mouse stained for CD4 or CD8 Ag or control Ab. Bottom, Brain from anti-Mig-treated mouse stained for CD4 Ag, CD8 Ag, or control Ab. Note the increased cellularity and staining for CD4 and CD8 Ag in the NRS-treated mouse as compared with the anti-Mig-treated mouse.

View this table:
Table II.

Decreased T lymphocyte infiltration into the CNS of anti-Mig-treated mice

Mig has been shown to exert a chemotactic effect on T cells through interaction with the receptor CXCR3 (10, 15, 16). Therefore, to determine whether CXCR3 levels were altered within the CNS of MHV-infected mice treated with anti-Mig, RT-PCR was performed on total RNA isolated from brains of mice from both groups at 7 days p.i. As seen in Fig. 5⇓, CXCR3 mRNA transcript levels within the CNS of mice treated with anti-Mig are markedly lower than levels present in the brains of mice treated with NRS. Similar to Mig, IP-10 has been shown to be chemotactic for CD4+ and CD8+ T lymphocytes through interaction with the receptor CXCR3. Determination of the levels of IP-10 at 7 days p.i. by RPA analysis revealed a decrease (∼18%) in IP-10 mRNA transcripts in the CNS of mice treated with anti-Mig antisera when compared with NRS control mice (data not shown).

FIGURE 5.
FIGURE 5.

Decreased levels of CXCR3 in brains of mice treated with anti-Mig. RT-PCR analysis of the effect of anti-Mig treatment on CXCR3 expression within the CNS of MHV-infected mice. Total RNA was extracted from the brains of MHV-infected mice treated with either anti-Mig or NRS at 7 days p.i. and subjected to RT-PCR analysis. Mice treated with anti-Mig show a marked decrease in the levels of CXCR3 in the CNS when compared with NRS-treated mice. Top, CXCR3; bottom, L32 control (Sham, n = 1; anti-Mig, n = 2; NRS, n = 2).

Cytokine expression in MHV-infected mice treated with anti-Mig

One mechanism by which T cells contribute to host defense against MHV infection of the CNS is through the release of the cytokine IFN-γ (2, 5, 6). To determine whether IFN-γ levels were altered in MHV-infected mice treated with anti-Mig, cytokine mRNA transcript levels within the CNS of anti-Mig- or NRS-treated mice were evaluated by RPA at days 3 and 7 p.i. Quantification of signal intensities indicated that at 3 days p.i. there were no appreciable differences in cytokine mRNA expression between mice treated with anti-Mig and NRS. In contrast, anti-Mig-treated mice displayed an 85% decrease in IFN-γ transcripts (p < 0.05) as well as a 65.1% decrease in IFN-β transcript levels (p < 0.02) as compared with mice treated with NRS at 7 days p.i. (Table III⇓). Consistent with the RPA results, IFN-γ protein levels were reduced by 91% (p < 0.05) within the CNS of anti-Mig-treated mice as compared with NRS-treated mice as determined by ELISA (Table IV⇓). Coinciding with decreased IFN-γ levels within anti-Mig-treated mice was a 4-fold increase (p < 0.02) in levels of the anti-inflammatory Th2 cytokine IL-10, whereas another Th2-associated cytokine, IL-4, was not detected in either group of mice at this time point (Table IV⇓). Treatment of mice with other anti-chemokine antisera such as anti-RANTES does not result in altered IL-10 levels, suggesting that the effect observed is specific for Mig neutralization (data not shown).

View this table:
Table III.

Cytokine mRNA levels

View this table:
Table IV.

Cytokine protein levels (ELISA)

Discussion

Mig is a member of the CXC subfamily of chemokines that has been shown to have multiple functions including contributing to an antitumor effect and inducing T cell and NK migration through interaction with CXCR3 (10, 15, 24, 32). More recent studies have implicated a role for Mig in host defense following viral infection (23, 26). Expression of Mig from a recombinant vaccinia virus protected nude mice following viral infection, whereas mice receiving a control vaccinia vector succumbed to the generalized infection (23). Successful clearance of virus by mice infected with the recombinant Mig virus was accompanied by a significant increase in mononuclear cell infiltration to sites of infection (23). Salazar-Mather et al. (26) have recently demonstrated that Mig expression is important in viral clearance in mice infected with murine cytomegalovirus, presumably through the recruitment of antiviral effector cells to sites of infection.

This study has evaluated the role of Mig in contributing to host defense following MHV infection of the CNS. The results presented demonstrate similarities as well as differences between Mig and IP-10 as they relate to MHV-induced CNS disease. Unlike IP-10, which is expressed during acute and chronic disease, expression of Mig was limited to the acute stage of infection (9). The highest levels of Mig mRNA transcripts were detected at day 7 p.i. followed by a 60% decrease by day 12 p.i. During this time, astrocytes were determined to be a cellular source of Mig; this is consistent with previous studies (33, 34). In contrast to IP-10, Mig transcripts are not detected at day 2 p.i., which represents a time in which very little IFN-γ is present within the CNS (Table III⇑). Therefore, given the dependence of Mig on IFN-γ as a triggering signal, this may explain why Mig is not seen at this stage of infection. The presence of IP-10 transcripts at day 2 is consistent with a previous study and can now be explained by data indicating IFN-β is present at this time (9). In addition, Mig transcripts were undetectable at day 35 p.i. at which point surviving animals were persistently infected with virus and undergoing chronic demyelination. Together, these results suggest that expression of Mig is important during the acute stage of disease but does not contribute to the pathology of chronic demyelination. In support of this is the demonstration that Ab-mediated neutralization of Mig activity resulted in a shift from a protective Th1 to a Th2 response characterized by 1) a significant reduction (p < 0.005) in CD4+ and CD8+ T cell infiltration into the CNS; 2) decreased IFN-γ expression (p ≤ 0.05); and 3) increased levels (p ≤ 0.02) of the anti-inflammatory Th2 cytokine IL-10 as compared with levels found in NRS-treated mice. This, in turn, correlated with a marked increase in mortality accompanied by a significant increase (p ≤ 0.001) in viral titers within the CNS of anti-Mig-treated mice vs NRS-treated mice.

The results presented support and extend an earlier study from our laboratory that determined that IP-10 expression is important in host defense against MHV infection of the CNS by contributing to mononuclear cell infiltration into the CNS and viral clearance (4). Collectively, these results indicate that both Mig and IP-10 are important contributors in host defense from MHV infection of the CNS by promoting a protective Th1 response. Due to the fact that Mig and IP-10 share receptor use and functional properties, it is interesting that neutralization of Mig did not result in an IP-10-mediated compensatory effect in host defense. However, this may be explained by the demonstration that anti-Mig-treated mice displayed decreased CNS transcript levels of IFN-γ and IFN-β, both of which are potent inducers of IP-10 expression (11, 12, 13). Therefore, it is possible that the decrease in stimulatory signals for IP-10 expression resulted in a diminished ability of IP-10 to compensate for the inhibition of Mig activity. Indeed, there was an ∼18% decrease in IP-10 transcript levels in mice treated with anti-Mig when compared with NRS-treated mice, which may help explain this observation.

The dramatic increase in CNS IL-10 levels within mice treated with anti-Mig is most likely a reflection of the reduced levels of IFN-γ present within these mice. Treatment of mice with anti-RANTES does not result in increased IL-10 levels, suggesting that the increase in IL-10 expression following anti-Mig treatment is not due to altered macrophage function (T.E.L. and M.T.L., unpublished observations). Furthermore, IL-10 expression has been shown to be dependent on the presence or absence of IFN-γ. Studies by Donnelly et al. (35) demonstrate that IFN-γ is capable of suppressing IL-10 expression in monocytes in a dose-dependent manner. In addition, in vitro infection of PBMC with human herpes virus type 6 results in increased expression of IL-10 that is inhibited following IFN-γ treatment. Neutralization of IFN-γ activity restored IL-10 expression by human herpes virus type 6-infected cells (36). Therefore, the increased levels of IL-10 may further diminish an effective host response to MHV by contributing to the inhibition of CNS inflammation as well as inhibiting cellular function as has been suggested to occur in experimental autoimmune encephalomyelitis and other models of CNS inflammation (37, 38, 39, 40, 41).

Although the reduction in IFN-γ levels was not surprising given the dramatic decrease in T cell infiltration, the lowered transcript levels of IFN-β was interesting. Neutralization of IP-10 does not affect IFN-β transcript levels within the CNS of MHV-infected mice, indicating that Mig may directly influence IFN-β expression (M.T.L. and T.E.L., unpublished observations). In addition, it is also possible that the reduction in IFN-β transcript levels in the CNS of anti-Mig-treated mice is a reflection of reduced cellular infiltration as has been suggested by Mahalingam et al. (23) in studies with a recombinant vaccinia virus expressing Mig. The increased viral burden within the CNS of mice treated with anti-Mig antisera is most likely the result of decreased levels of IFN-γ and IFN-β as both cytokines are considered to contribute to host defense against MHV infection (5, 6, 42).

In conclusion, the data presented in this study provide further evidence that chemokine expression is important in host defense against viral infection of the CNS by helping to promote a protective Th1 response (4). Moreover, these studies provide additional support for the concept that selected Ab-mediated targeting of chemokines is a powerful method of modulating the severity of neuroinflammation following viral infection (1, 4, 43, 44). Therefore, the use of Ab-mediated neutralization of chemokine activity may also provide attractive strategies for ameliorating the severity of human neuroinflammatory diseases such as multiple sclerosis in which chemokines have been postulated to have an important role in the pathology of disease (45, 46).

Acknowledgments

We thank Matthew Trifilo and William Glass for reading the manuscript and helpful discussion.

Footnotes

  • ↵1 This work was supported by National Multiple Sclerosis Society Research Grant RG 30393A1/T and National Institutes of Health Grants NS37336-01 (to T.E.L.) and CA39621 (to T.A.H.). M.T.L. is supported by National Institutes of Health Training Grant T32NS07444.

  • ↵2 Address correspondence and reprint requests to Dr. Thomas E. Lane, Department of Molecular Biology and Biochemistry, University of California, Irvine, 3205 Biological Sciences II, Irvine, CA 92697-3900. E-mail address: tlane{at}uci.edu

  • ↵3 Abbreviations used in this paper: MHV, mouse hepatitis virus; Mig, monokine induced by IFN-γ; IP-10, IFN-γ-inducible protein 10; NRS, normal rabbit serum; RPA, RNase protection assay; p.i., postinfection; CXCR3, CXC chemokine receptor 3; GFAP, glial fibrillary acidic protein.

  • Received July 24, 2000.
  • Accepted November 2, 2000.

References

  1. ↵
    Lane, T. E., M. T. Liu, B. P. Chen, V. C. Asensio, R. M. Samawi, A. D. Paoletti, I. L. Campbell, S. L. Kunker, H. S. Fox, M. J. Buchmeier. 2000. A central role for CD4+ T cells and RANTES in virus-induced central nervous system inflammation and demyelination. J. Virol. 74: 1415
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Pearce, B. D., M. V. Hobbs, T. S. McGraw, M. J. Buchmeier. 1994. Cytokine induction during T-cell-mediated clearance of mouse hepatitis virus from neurons in vivo. J. Virol. 68: 5483
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Williamson, J. S. P., S. A. Stohlman. 1990. Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells. J. Virol. 64: 4589
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Liu, M. T., B. P. Chen, P. Oertel, M. J. Buchmeier, D. Armstrong, T. A. Hamilton, T. E. Lane. 2000. Cutting edge: the T cell chemoattractant IFN-inducible protein 10 (IP-10) is essential in host defense against viral-induced neurologic disease. J. Immunol. 165: 2327
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Lane, T. E., A. D. Paoletti, M. J. Buchmeier. 1997. Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus. J. Virol. 71: 2202
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Parra, B., D. R. Hinton, N. W. Marten, C. C. Bergmann, M. T. Lin, C. S. Yang, S. A. Stohlman. 1999. IFN-γ is required for viral clearance from central nervous system oligodendroglia. J. Immunol. 162: 1641
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Luster, A. D.. 1998. Chemokines: chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338: 436
    OpenUrlCrossRefPubMed
  8. ↵
    Baggiolini, M., B. Dewald, B. Moser. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15: 675
    OpenUrlCrossRefPubMed
  9. ↵
    Lane, T. E., V. C. Asensio, N. Yu, A. D. Paoletti, I. L. Campbell, M. J. Buchmeier. 1998. Dynamic regulation of α and β chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease. J. Immunol. 160: 970
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Farber, J. M.. 1997. Mig and IP-10: CXC chemokines that target lymphocytes. J. Leukocyte Biol. 61: 246
    OpenUrlAbstract
  11. ↵
    Luster, A. D., J. C. Unkeless, J. V. Ravetch. 1985. γ-interferon transcriptionally regulates an early response gene containing homology to platelet proteins. Nature 315: 672
    OpenUrlCrossRefPubMed
  12. ↵
    Farber, J. M.. 1993. HuMIG: a new human member of the chemokine family of cytokines. Biochem. Biophys. Res. Commun. 192: 223
    OpenUrlCrossRefPubMed
  13. ↵
    Vanguri, P., J. M. Farber. 1990. Identification of CRG-2: an interferon-inducible mRNA predicted to encode a murine monokine. J. Biol. Chem. 265: 15049
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Farber, J. M.. 1990. A macrophage mRNA selectively induced by γ-interferon encodes a member of the platelet factor 4 family of cytokines. Proc. Natl. Acad. Sci. USA 87: 5238
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Loetscher, M., B. Gerber, P. Loetscher, S. A. Jones, L. Piali, I. C. Lewis, M. Baggiolini, B. Moser. 1996. Chemokine receptor specific for IP-10 and Mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 184: 963
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Piali, L., C. Weber, G. LaRosa, C. R. Mackay, T. A. Springer, I. Clark-Lewis, B. Moser. 1998. The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP-10 and Mig. Eur. J. Immunol. 28: 961
    OpenUrlCrossRefPubMed
  17. ↵
    Biddison, W. E., W. W. Cruikshank, D. M. Center, C. M. Pelfrey, D. D. Taub, R. V. Turner. 1998. CD8+ myelin peptide-specific T cells can chemoattract CD4+ myelin peptide-specific T cells: importance of IFN-inducible protein 10. J. Immunol. 160: 444
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Engelhardt, E., A. Toksoy, M. Goebler, S. Debus, E. B. Brocker, R. Gillitzer. 1998. Chemokines IL-8, GRO-α, MCP-1, IP-10 and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing. Am. J. Pathol. 153: 1849
    OpenUrlCrossRefPubMed
  19. ↵
    Bradley, L. M., V. C. Asensio, L. K. Schoitz, J. Harbertson, T. Krahl, G. Patstone, N. Woolf, I. L. Campbell, N. Sarvetnick. 1999. Islet-specific Th1 but not Th2, cells secrete multiple chemokines and promote rapid induction of autoimmune diabetes. J. Immunol. 162: 2511
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Shields, P. L., C. M. Morland, M. Salmon, S. Qin, S. G. Hubscher, D. H. Adams. 1999. Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver. J. Immunol. 163: 6236
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Romagnani, P., C. Beltrame, F. Annunziato, L. Lasagni, M. Luconi, G. Galli, L. Cosmi, E. Maggi, M. Salvadori, C. Pupilli, M. Serio. 1999. Role for interactions between IP-10/Mig and CXCR3 in proliferative glomerulonephritis. J. Am. Soc. Nephrol. 10: 2518
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Glabinski, A. R., M. Tani, R. M. Strieter, V. K. Tuohy, R. M. Ransohoff. 1997. Synchronous synthesis of α and β chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis. Am. J. Pathol. 150: 617
    OpenUrlPubMed
  23. ↵
    Mahalingam, S., J. M. Farber, G. Karupiah. 1998. The interferon-inducible chemokines MuMig and Crg-2 exhibit antiviral activity in vivo. J. Virol. 73: 1479
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Tannenbaum, C. S., R. Tubbs, D. Armstrong, J. H. Finke, R. M. Bukowski, T. A. Hamilton. 1998. The CXC chemokines IP-10 and Mig are necessary for IL-12-mediated regression of the mouse RENCA tumor. J. Immunol. 161: 927
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Keane, M. P., J. A. Belperio, D. A. Arenberg, M. D. Burdick, Z. J. Xu, Y. Y. Xue, R. M. Strieter. 1999. IFN-γ-inducible protein-10 attenuates bleomycin-induced pulmonary fibrosis via inhibition of angiogenesis. J. Immunol. 163: 5686
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Salazar-Mather, T. P., T. A. Hamilton, C. A. Biron. 2000. A chemokine to cytokine to chemokine cascade critical in antiviral defense. J. Clin. Invest. 105: 985
    OpenUrlCrossRefPubMed
  27. ↵
    Dalziel, R. G., P. W. Lampert, M. J. Buchmeier. 1986. Site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein E2 results in reduced neurovirulence. J. Virol. 59: 463
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Hirano, N., T. Murakami, K. Fujiwara, M. Matsumoto. 1978. Utility of the mouse cell line DBT for propagation and assay of mouse hepatitis virus. Jpn. J. Exp. Med. 48: 71
    OpenUrlPubMed
  29. ↵
    Houtman, J. J., J. O. Fleming. 1996. Pathogenesis of mouse hepatitis virus-induced demyelination. J. Neurovirol. 2: 361
    OpenUrlCrossRefPubMed
  30. ↵
    Fazakerly, J. K., S. E. Parker, F. Bloom, M. J. Buchmeier. 1992. The V5A13.1 envelope glycoprotein deletion mutant of mouse hepatitis virus type-4 is neuroattenuated by its reduced rate of spread in the central nervous system. Virology 187: 178
    OpenUrlCrossRefPubMed
  31. ↵
    Buchmeier, M. J., T. E. Lane. 1999. Viral-induced neurodegenerative disease. Curr. Opin. Microbiol. 2: 398
    OpenUrlCrossRefPubMed
  32. ↵
    Sgadari, C., J. M. Farber, A. L. Angiolillo, F. Liao, J. Teruya-Feldstein, P. R. Burd, L. Yao, G. Gupta, C. Kanegane, G. Tosato. 1997. Mig, the monokine induced by interferon-γ, promotes tumor necrosis in vivo. Blood 89: 2635
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Simpson, J. E., J. Newcomb, M. L. Cuzner, M. N. Woodroofe. 2000. Expression of the interferon-γ-inducible chemokines IP-10 and Mig and their receptor, CXCR3, in multiple sclerosis lesions. Neuropathol. Appl. Neurobiol. 26: 133
    OpenUrlCrossRefPubMed
  34. ↵
    Vanguri, P.. 1995. Interferon-γ inducible genes in primary glial cells of the central nervous system: comparisons of astrocytes with microglia and Lewis with brown Norway rats. J. Neuroimmunol. 56: 35
    OpenUrlCrossRefPubMed
  35. ↵
    Donnelly, R. P., S. L. Freeman, M. P. Hayes. 1995. Inhibition of IL-10 expression by IFN-γ up-regulates transcription of the TNF-α in human monocytes. J. Immunol. 155: 1420
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Arena, A., M. C. Liberto, D. Iannello, A. B. Capozza, A. Foca. 1999. Altered cytokine production after human herpes virus type 6 infection. New Microbiol. 22: 293
    OpenUrlPubMed
  37. ↵
    Groux, J., F. Cottrez, M. Rouleau, S. Mauze, S. Antonenko, S. Jurst, T. Mcneil, M. Bigler, M. G. Romcarolo, R. L. Coffman. 1999. A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigen-presenting cells. J. Immunol. 162: 1723
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Samoilova, E. B., J. L. Horton, Y. Chen. 1998. Acceleration of experimental autoimmune encephalomyelitis in interleukin-10-deficient mice: roles of interleukin-10 in disease progression and recovery. Cell. Immunol. 188: 118
    OpenUrlCrossRefPubMed
  39. ↵
    Knoblach, S. M., A. I. Faden. 1998. Interleukin-10 improves outcome and alters proinflammatory cytokine expression after experimental traumatic brain injury. Exp. Neurol. 153: 143
    OpenUrlCrossRefPubMed
  40. ↵
    Issazadeh, S., J. C. Lorentzen, M. I. Mustafa, B. Hojeberg, A. Mussemer, T. Olsson. 1996. Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 in transforming growth factor-β. J. Neuroimmunol. 69: 103
    OpenUrlCrossRefPubMed
  41. ↵
    Brewer, K. L., J. R. Betehea, R. P. Yezierski. 1999. Neuroprotective effects of interleukin-10 following excitotoxic spinal cord injury. Exp. Neurol. 159: 484
    OpenUrlCrossRefPubMed
  42. ↵
    Minagawa, H., A. Takenaka, S. Mohri, R. Mori. 1987. Protective effect of recombinant murine interferon-β against mouse hepatitis virus infection. Antiviral Res. 8: 85
    OpenUrlCrossRefPubMed
  43. ↵
    Karpus, W. J., N. W. Lukacs, B. L. McRae, R. M. Strieter, S. L. Kunkel, S. D. Miller. 1995. An important role for the chemokine macrophage inflammatory protein-1 α in the pathogenesis of the T cell mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155: 5003
    OpenUrlAbstract
  44. ↵
    Kennedy, K. J., R. M. Stricter, S. L. Kunkel, N. W. Lukacs, W. J. Karpus. 1998. Acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the CC chemokines macrophage inflammatory protein-1 α and monocyte chemotactic protein-1. J. Neuroimmunol. 92: 98
    OpenUrlCrossRefPubMed
  45. ↵
    Sorensen, T. L., M. Tani, J. Jensen, V. Pierce, C. Lucchinetti, V. A. Folcik, S. Qin, J. Rottman, F. Sellebjerg, R. M. Strieter, et al 1999. Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J. Clin. Invest. 103: 807
    OpenUrlCrossRefPubMed
  46. ↵
    Hvas, J., C. McLean, J. Justesen, G. Kannaourakis, L. Steinman, J. R. Oksenberg, C. C. Bernard. 1997. Perivascular T cells express the pro-inflammatory chemokine RANTES mRNA in multiple sclerosis lesions. Scand. J. Immunol. 46: 195
    OpenUrlCrossRefPubMed
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