INTRODUCTION (Wesselingh et al., 1994) .
Variations between viral infections resulting in CNS The goal of the immune response during viral infection inflammation prompted an examination of the temporal is to limit replication via induction of both nonspecific and induction of CNS cytokines during fatal and nonfatal CNS specific antiviral effectors. Acute viral infections of the ceninfections by variants of the JHM strain (JHMV) of mouse tral nervous system (CNS) result in vigorous, but in some hepatitis virus (MHV). In immunocompromised hosts instances limited, host immune responses (Sedgwick and JHMV replicates unchecked in the CNS demonstrating Dorries, 1991) . In contrast to responses in the periphery the importance of immune effectors in limiting CNS virus where limiting virus replication can generally be carried replication ; Houtman and out with minimal regard to tissue damage, within the CNS Fleming, 1996b; . Effector checks and balances minimize inflammatory-mediated mechanisms implicated in protection and clearance of damage while limiting viral-induced cytopathology. Al-JHMV from the CNS include cell-mediated immunity and though a wide range of immune effectors are often induced, both neutralizing and nonneutralizing antibodies. JHMV predominant anti-viral mechanisms appear related to the provides an interesting paradigm of acute viral encephapathogenesis strategy of the individual agent. For example, litis not only because of its associated demyelination infection of mice with lymphocytic choriomeningitis virus (Weiner, 1973; Lampert et al., 1973) but also because induces a predominant CD8 / cytotoxic T lymphocyte (CTL) some immune effector mechanisms prevent death via response (Lehmann et al., 1988) . By contrast, resolution of directly reducing CNS virus replication while other immeasles virus-encephalitis in mice is mediated by CD4 / T mune effectors prevent death without significantly altercells and correlates with the local production of IFN-g ing virus replication Hout-(Finke et al., 1995) . Finally, resolution of Sindbis virus-inman and Fleming, 1996b; . A duced encephalitis is related to induction of neutralizing common theme appears to be prevention of neuronal infection by reducing viral load or preventing neuronal infection, most likely via cytokines.
tion and clearance of JHMV from the CNS are not yet and rarely neurons (2.2v-1). These viruses contrast to the predominantly neuronotropic OBLV-60 variant previously clear. The antiviral effects of CD8 / T cells appear to be due to direct lysis of infected cells; however, CD8 / and examined (Pearce et al., 1994; . CD4 / T cells may also exert antiviral activity indirectly MATERIAL AND METHODS via cytokine secretion (Biron, 1994) . Neither infected neurons nor oligodendrocytes appear susceptible to major Mice and viruses histocompatibility (MHC) class I-mediated killing in vivo, C57BL/6 mice were purchased from the Jackson Laboconsistent with the inability of JHMV-specific CTL to clear ratory (Bar Harbor, ME) at 6 weeks and maintained in virus from infected oligodendroglia (Stohlman et al., the University of Southern California vivarium. All mice 1995b). Furthermore, clearance of JHMV from the CNS were used at 7 weeks of age. To produce a lethal infecis inhibited, but not abolished, in mice genetically defition, mice were infected by intracerebral inoculation (i.c.) cient in perforin-mediated cytolysis (Lin et al., 1997) .
with 100 PFU of the plaque-purified DM isolate of JHMV These data suggest the possibility that cytokines contrib- (Stohlman et al., 1982) in a volume of 32 ml. This virus has ute to either clearance or protection from JHMV infection. the plaque size and pathogenesis similar to the parental During JHMV infection of the CNS there is an abrupt suckling mouse brain pool of JHMV originally described increase in mRNA encoding interleukin-1 (a and b), ILby Weiner (1973) and produces a lethal encephalomyeli-6, tumor necrosis factor (TNF)-a, and interferon (IFN)-g, tis with minimal demyelination apparent at the time of at the time of maximal decrease in virus replication and death. To produce a sublethal infection, mice were inmononuclear cell infiltration (Pearce et al., 1994) . No IFNfected with 25 PFU of the 2.2v-1 monoclonal antibodyg mRNA was detected in immunodeficient mice, sugderived neutralization-resistant variant of JHMV (Fleming gesting this cytokine may be important during viral clearet al., 1986) . This variant replicates predominantly in oliance (Pearce et al., 1994) . Consistent with this concept, godendroglia producing a flaccid paralysis. Although vimice treated with anti-IFN-g are more susceptible to ral antigen is cleared from survivors by 30 days postinfec-JHMV, while administration of IFN-g provides protection tion (p.i.), viral RNA persists for at least 12 months (Adami (Smith et al., 1991) . IL-6, TNF-a, and type 2 nitric oxide et al., 1995) . Groups of at least 3 mice were sacrificed synthase (iNOS) have also been detected in the CNS at various times p.i. Immunosuppression was induced during acute JHMV infection (Sun et al., 1995; by lethal irradiation (850R) 24 hr prior to infection. Shamet al., 1995a; while IL-1b, IL-6, TNF-a, infected mice were injected i.c. with 32 ml of sterile endoand iNOS were detected in the CNS of chronically intoxin-free phosphate-buffered saline (PBS). fected mice (Sun et al., 1995) . The complex interactions of multiple immune effector mechanisms during JHMV Virus titration infection may reflect both the relative immune privilege Virus titers were determined by plaque assay using of the CNS (Sedgwick and Dorries, 1991) as well as the monolayers of DBT cells as previously described (Stohlspecific tropism of the virus for CNS cell types. Neuroman et al., 1982) . One-half of the brain was homogenized tropic MHV isolates differ in tropism and include viruses using Tenbrock tissue homogenizers in 2.0 ml of Dulbecwith predominant tropisms for astrocytes, microglia, and co's PBS, pH 7.4. The remaining half was taken for histooligodendroglia as well as neurons (Fleming et al., 1986;  pathology or RNA extraction (see below). Following cen- Perlman and Reis, 1987; Kyuwa and Stohlman, 1990;  trifugation at 1500 g for 7 min at 4Њ, supernatants were Pearce et al., 1994; Stohlman et al., 1995a) . The balance assayed immediately or frozen at 070Њ. Data presented between limiting viral replication and preserving CNS are the average titer of groups of three or more mice. function occasionally results in incomplete viral clearance and a persistent CNS infection which may or may Antibody titration not involve the continued presence of infectious virus JHMV-specific IgM, IgG1, and IgG2a antibodies were quantitated by ELISA as previously described (Lin et al., 1996b) . Persistence of infectious virus correlates with the 1997) using rabbit anti-mouse IgM, IgG1, or IgG2a antipresence of CTL escape variants (Pewe et al., 1996) .
bodies (Cappel, Costa Mesa, CA). Concentrations of se-To understand the complex interrelationships between rum antibodies were expressed as the highest dilution encephalitis, protection, and viral clearance leading to a with O.D. values three times above background level. persistent infection of the CNS, the expression of pro-Neutralizing antibodies were tested in serum as preand anti-inflammatory cytokine mRNAs in the CNS of viously described (Lin et al., 1997) . mice undergoing either lethal or sublethal JHMV infection were compared. The two JHMV chosen for study infect Histology either primarily microglia and astrocytes, less frequently oligodendroglia and neurons (DM) or primarily oligoden-Histopathologic analysis was performed as previously described (Stohlman et al., 1995a) . Briefly, tissues were droglia, much less frequently microglia and astrocytes, fixed for 3 hr in Clark's solution (75% ethanol, 25% glacial with 32 P-ATP-labeled internal oligonucleotide probes. Membranes were washed (three times; 21 SSC, 0.1% acidic acid) and embedded in paraffin. Sections were stained with hematoxylin and eosin or luxol fast blue. Distri-SDS; room temperature), exposed to Storage Phosphor Screens (Molecular Dynamics, Sunnyvale, CA), and bution of JHMV antigen was examined by immunoperoxidase staining (Vectastain-ABC kit; Vector Laboratory, Burlin-scanned using a phosphorimaging scanner (Molecular Dynamics). game, CA) using the anti-JHMV mAb J3.3 specific for the viral nucleocapsid protein (Fleming et al., 1983) .
Radioactive signals of cytokine cDNA were quantified and normalized to the house-keeping enzyme hypoxanthine phosphoriboxyltransferase (HPRT) values to adjust Cytokine mRNA expression for differences in cDNA as previously described (Cua et al., 1995 (Cua et al., , 1996 . The sample with the highest specific Brains were processed individually to prevent contamination. RNA was isolated from half brains by homogeni-activity was designated the 100% maximal response and values for the remainder were derived as percentage of zation at room temperature in guanidinium isothiocyanate using Tenbrock tissue homogenizers as previously the highest value. Data shown are mean values for 3-4 mice at each time point { 1 standard deviation. described (Cua et al., 1995) . Samples were sheared prior to centrifugation through 5.4 M cesium chloride at 100,000 g for 18 hr to isolate RNA. The cDNA were pre-RESULTS pared using avian myeloblastosis reverse transcriptase Acute and subacute JHMV-induced encephalitis (Promega, Madison, WI) and oligo dT primers (Promega) for 60 min at 42Њ. Expression of cytokine mRNA was Fatal encephalomyelitis induced by JHMV is associated with minimal demyelination (Kyuwa and Stohlman, determined by semiquantitative PCR analysis, following procedures previously described (Cua et al., 1995 (Cua et al., , 1996 (Cua et al., ). 1990 Houtman and Fleming, 1996b) . This contrasts with infection by 2.2v-1 which produces an acute nonfatal en-PCR was performed using AmpliTaq DNA polymerase (Perkin-Elmer, Branchburg, NJ) and specific cytokine cephalomyelitis with extensive demyelination (Fleming et al., 1986; Wang et al., 1990) . Although both viruses primers for IFN-g, IL-1a, IL-1b, IL-4, IL-6, IL-10, TNF-a (Murphy et al., 1993; Cua et al., 1996) , and IL-12p40. The replicated rapidly to high titer in the CNS (Fig. 1A) , JHMVinfected mice succumbed within 8 days while 2.2v-1-sequences of the IL-12p40 oligonucleotides primers and probe used are as follows: 5 primer, GAC CCT GCC infected mice underwent a subacute disease with little or no mortality (Fig. 1B) . Peak 2.2v-1 replication was at CAT TGA ACT GGC; 3 primer, CAA CGT TGC ATC CTA GGA TCG; oligoprobe, TGT CTG CGT GCA AGC TCA Day 3 while the peak of JHMV replication was delayed until Day 5. 2.2v-1 clearance began at day 5 p.i. and by GGA. Amplification was carried out using 35 cycles of one denaturation step at 94Њ (45 sec), primer annealing Day 7 virus was nearly undetectable. By contrast, titers in JHMV-infected mice initially decreased at Day 7 p.i. at 59Њ (45 sec), extension step at 72Њ (1.5 min), followed by a final extension step for 7 min. For IL-4 and iNOS a and detectable virus was still present in the CNS of moribound mice at Day 8 p.i. (Fig. 1A ). During lethal JHMV nested PCR was performed by using internal primers in a second round of PCR (25 cycles) under the conditions infection, virus replication within the CNS is not reduced as rapidly as in mice which survive infection ( Fig. 1A ) described above. The oligonucleotide primers used in the second PCR for IL-4 were the corresponding se-consistent with the notion that rapid clearance correlates positively with protection. Consistent with these findings, quences for the 5-primer and the probe described by Cua et al. (1996) . The nucleotide sequence for the IL-4 immunohistologic examination of the brains of JHMVinfected mice at Day 7 showed abundant viral antigen oligonucleotide probe was TTG AAG GAG GTC ACA GGA GAA GGGA (Sideras et al., 1987) . The 5 and 3 outer in regions of encephalitis while only focal residual viral antigen was found in 2.2v-1-infected animals (Fig. 2) . En-primer sequences for iNOS were GCC TTC CGC AGC TGG GCT GT and ATG TGG TAG CCA CAT CCC GAG cephalitis was prominent in mice infected with either 2.2v-1 or JHMV and no differences in the amount or distri-CC, respectively (Lyons et al., 1992) . Internal 5 and 3 iNOS primers were AGC TAC TGG GTC AAA GAC AAG bution of mononuclear cell infiltrates were found at Day 7 (Fig. 2) . No serum neutralizing antibodies were de-AGG CT and the 3 outer primer, respectively. The oligonucleotide probe consisted of the sequence CTC CCT tected in either group by Day 9 postinfection, even though the virus titer in the CNS had declined over 3 log 10 (Lin TCC GAA GTT TCT GGC AGC A.
For quantification, PCR products were diluted in dena-et al., 1997; data not shown). In contrast to neutralizing antibodies, IgM was first detected at Day 5 post-2.2v-1 turing solution (0.4 N NaOH, 25 mM EDTA), neutralized with Tris-HCI (1.0 M; pH 8.0), and transferred to 0.45 infection (data not shown) and both IgG1 and IgG2a were detected as early as 7 days p.i. (Fig. 1C) . The IgG1 and mm Nytran membranes (Schleicher & Schuell, Keene, NH) using a Minifold I dot blot apparatus (Schleicher & IgG2a response suggest the absence of a shift toward either a Th1-or Th2-type response reported to be in-Schuell). Membranes were hybridized (60Њ; overnight) volved in the response to Sindbis virus-induced encepha-deficient mice, which showed no evidence of IFN-g mRNA, suggests TNF-a may also contribute to iNOS litis (Wesselingh et al., 1994) . mRNA induction (Colasanti et al., 1995; Gazzinelli et al., 1993) . Consistent with this notion, TNF-a mRNA was first Proinflammatory cytokines detected at Day 3 in mice undergoing a lethal infection The mRNA encoding IFN-g increased in both groups and at Day 5 in mice sublethally infected (Fig. 3C ). Similar of mice through Day 5 postinfection, consistent with the to the kinetics of IFN-g, TNF-a mRNA increased until rapid accumulation of both NK and T cells in the CNS of death of lethally infected mice. In mice undergoing a infected mice (Williamson et al., 1991; Williamson, 1992) sublethal infection, TNF-a mRNA declined following the (Fig. 3A) . No IFN-g mRNA was detected in either shampeak of virus replication and approached baseline levels infected mice or in infected immunodeficient mice. Durby 14 days p.i. ing the lethal JHMV infection IFN-g mRNA did not in-Similar to both TNF-a and iNOS, IL-12 is secreted from crease between Day 5 and Day 7. However, in mice macrophages during the induction of cell-mediated imundergoing a sublethal infection the level of IFN-g mRNA munity and protects from a number of viral infections continued to increase to Day 7 and remained elevated, via a IFN-g-dependent mechanism (Ozmen et al., 1995;  suggesting the possibility that IFN-g is important follow- Orange and Biron, 1996) . No IL-12 mRNA was found foling infection with a JHMV variant tropic for oligodendroglowing sham infection; however, IL-12 mRNA increased lia. Even though IFN-g mRNA increased during the early rapidly and peaked at 3 days following both infections phase of infection, a sharp transient increase in iNOS (Fig. 4A ). Increased IL-12 mRNA also occurred in immu-mRNA was detected at Day 5 p.i. in mice with a lethal nodeficient mice at 3 days p.i., suggesting a direct reencephalomyelitis (Fig. 3B) . Only a slight increase was sponse to infection which may be related to the recently detected in mice undergoing subacute encephalomyelidescribed IFN-g-independent induction of IL-12 (Heinzel tis. Interestingly, infection of immunodeficient mice with et al., 1996) . IL-12 mRNA levels decreased after Day 3 JHMV induced the accumulation of iNOS mRNA to apand nearly approached base line levels found in uninproximately 50% the level found in infected immunocomfected mice by 14 days p.i. petent mice, suggesting a direct response to viral infec-The IL-1b mRNA level found at Day 1 p.i. declined by 3 days p.i., consistent with induction of an early transient tion. The increase in iNOS and TNF-a mRNA in immuno- increase in IL-1b mRNA in sham-infected mice (Fig. 4B ).
subacute infection (Fig. 4C ) and then declined but never returned to baseline. In lethally infected mice the peak IL-1b mRNA peaked at Day 5 following sublethal infection and subsequently declined as virus was cleared of IL-1a mRNA was delayed (Day 5 p.i.) and then declined as the animals succumbed to infection (Fig. 4C ). IL-6 from the CNS. Following a lethal infection, the quantity of IL-1b mRNA increased from Day 3 p.i. until death. IL-mRNA peaked at Day 5 postinfection in lethally infected mice and declined by Day 7 as virus was cleared from 1a mRNA peaked at Day 3 p.i. in the mice undergoing a the CNS (Fig. 4D) . In contrast to the lethal infection, the of IL-4 mRNA expression following acute and subacute infections showed that the levels increased in parallel levels of IL-6 mRNA increased rapidly and peaked at Day 3 p.i. following subacute infection. The level then through Day 7 p.i. (Fig. 5A ). In 2.2v-1-infected mice, the level of IL-4 mRNA continued to increase until Day 9 p.i. declined rapidly by Day 5 and had reached baseline levels by Day 9 p.i. No IL-6 mRNA was detected in sham-and then declined slightly by Day 14. infected mice, suggesting a rapid response to virus infection. Very low levels of IL-6 mRNA were detected in im-DISCUSSION munodeficient mice infected with either virus.
JHMV produces an acute CNS infection associated with several immune effector mechanisms, including Th2-related cytokines both CD4 / and CD8 / T cells Houtman and Fleming, 1996b) . Kinetic analysis of cellular IgG1 and IgG2a virus-specific antibodies were detected in survivors of JHMV infection; however, there appeared CNS infiltrations during JHMV infection of mice shows that NK cells accumulate prior to CD8 / T cells, which in to be little relationship between induction of antibody and control of JHMV infection within the CNS. Induction of both turn precede accumulation of CD4 / T cells and macrophages (Williamson et al., 1991; Williamson, 1992) . There isotypes suggest that Th1 and Th2 cytokines are induced by JHMV infection. The kinetics of IL-10 mRNA induction is no direct evidence for a role of NK cells in suppressing JHMV replication (Houtman and Fleming, 1996a) ; how-was of interest due to the association of IL-10 with reduced Th1 activity in vitro and with remission during exper-ever, CD8 / CTL appear to be critical immune effectors (Williamson and Stohlman, 1990; Stohlman et al., 1995b) . imental allergic encephalomyelitis (Kennedy et al., 1992) . IL-10 mRNA was first detected at Day 3 p.i. in lethally Recent analysis of JHMV pathogenesis in mice deficient in perforin suggests that in addition to cytolytic effectors infected mice, but not until Day 5 postinfection in the CNS of the mice undergoing a subacute encephalitis. However, other immune components also contribute to sterilizing immunity (Lin et al., 1997) . Similarly, the adoptive transfer at the time most lethally infected mice were about to succumb to infection (Day 7), there was no difference in the of virus-specific CD4 / T cells to JHMV-infected mice demonstrates that some clones protect via reducing viral peak levels of IL-10 mRNA between the two groups. The kinetics of IL-10 mRNA accumulation differed between the replication (Yamaguchi et al., 1991) , while others protect without reducing virus replication , groups; IL-10 mRNA accumulation in mice undergoing a sublethal infection was slower and remained at peak lev-suggesting that cytokines may play an important role in providing sterile immunity. els until Day 9 p.i., prior to declining to near basal levels by Day 14. No IL10 mRNA was detected in the CNS of In general the kinetics of cytokine mRNA expression correlated with the temporal presence of CNS infiltrating sham-infected or infected immunodeficient mice. No IL-4 mRNA was detected following a single amplification dur-mononuclear cells. Many cytokine transcripts, with the exceptions of IL-12, IL-1a, and IL-6, were maximally ex-ing lethal or sublethal JHMV infections. However, after a second amplification, low abundant mRNAs were de-pressed by 7 day p.i., near the peak inflammatory cell infiltration and during the elimination of virus from the tected (Fig. 5A) . No IL-4 mRNA was detected in either sham-infected or infected immunosuppressed mice fol-CNS (Williamson et al., 1991; Williamson, 1992) . Previous data using the OBLV-60 JHMV variant which has a selec-lowing two amplifications (data not shown). The kinetics tive tropism for neurons suggested a correlation between increasing time following subacute infection, consistent with the resolution of encephalitis. It is interesting that IFN-g induction, T cell accumulation, and reduction of virus replication (Pearce et al., 1994) . The semiquantita-the CNS of mice with active macrophage-mediated demyelination (Day 14 p.i.) showed little evidence of TNF-tive kinetic analysis of IFN-g mRNA in the CNS of mice undergoing both lethal and sublethal JHMV infections a mRNA, consistent with the inability of anti-TNF-a to prevent JHMV-mediated demyelination (Stohlman et al., supports the positive correlation between IFN-g and viral clearance. However, the OBLV-60 JHMV variant is 1995a). A surprising number of mRNAs peaked relatively early cleared from the CNS of IFN-g-deficient mice , consistent with IFN-g exhibiting poor in vitro following JHMV infection. The mRNAs encoding iNOS, IL-12, IL-1a, IL-1b, and IL-6 peaked either prior to or anti-JHMV activity (Zhang et al., 1997) and inability of rIFN-g to inhibit CNS virus replication (Smith et al., 1991) . coincident with initiation of viral clearance. In most cases (except iNOS mRNA) the levels were either higher or These data contrast with other viral-induced encephalopathies in which IFN-g plays a significant role (Kundig et increased more rapidly in the mice undergoing subacute infections. Accumulation of iNOS mRNA was first de-al., 1993; Finke et al., 1995) , including some (Yu et al., 1996) , but not all (Wesselingh et al., 1994) , neuronotropic tected in mice undergoing a lethal infection coincident with the initial detection of IFN-g mRNA. However, the viruses. The kinetics of IFN-g mRNA induction suggests that it may play a more prominent role in the pathogene-mRNA levels declined as virus replication declined, suggesting a direct effect of virus on iNOS induction. In sis of JHMV variants with predominant tropisms for microglia, astrocytes (JHMV), or oligodendroglia (2.2v-1).
contrast to lethal infections, iNOS mRNA lagged detection of IFN-g in mice undergoing subacute infections and The isotype diversity of the anti-JHMV antibody response suggests that both Th1 and Th2 subsets of CD4 / increased to less than 50% the level detected in mice undergoing a lethal infection. Similar to the recent data T cells are activated during infection. IL-4 mRNA accumulation in the CNS corresponds to infiltration of Th2 cells demonstrating low levels of iNOS in the CNS of both nude mice and mice deficient in IFN-g , (Cua et al., 1995) and kinetic analysis suggests that T cells expressing Th2 cytokine profiles are recruited into iNOS mRNA in immunodeficient mice was approximately 50% the levels detected in the CNS of intact mice at Day the CNS with nearly equal kinetics in both lethally and sublethally infected mice (Fig. 5A) . IL-4 increases the 3 p.i. Although JHMV is susceptible to inhibition by iNOS in vitro, iNOS is not associated with in vivo protection severity of encephalitis (Ikemoto et al., 1995) and could potentially play a role in JHMV persistence via inhibition . IL-12, predominantly produced by cells of the myelo-of viral clearance (Moran et al., 1996) . In support of the recruitment of Th2 cells, IL-10 mRNA also increased with monocytic lineage, is associated with the induction of Th1 CD4 / T cells (Brunda, 1994) . IL-12 mRNA peaked kinetics similar to those of IFN-g and IL-4. Whether this difference in detection of Th2 cytokines is due to differ-early (Day 3) in mice undergoing both lethal and sublethal JHMV infections. However, no significant differences ences in mouse strains or the selective tropism of the virus is not known. It is interesting that although IL-10 is were found comparing mRNA levels in immunodeficient mice to intact mice. This may suggest that JHMV infection secreted by activated microglia in vitro (Lodge and Sriram, 1996) , no IL-10 mRNA was detected in sham-induces transcription of IL-12 mRNA in CNS cells. In addition, 2.2v-1 infects predominantly, but not exclu-infected or immunodeficient mice. This contrasts with other cytokine mRNA detected in either sham-infected sively, oligodendroglia, while JHMV infects predominantly microglia and astrocytes. The relatively higher or virus-infected immunodeficient hosts (see below).
TNF-a mRNA is induced following JHMV infection level of IL-12 mRNA in 2.2v-1-infected mice suggests the possibility that oligodendroglia transcribe IL-12 mRNA in (Pearce et al., 1994; Stohlman et al., 1995a; Sun et al., 1995) and TNF-a is present during both the acute and response to JHMV infection, similar to the induction of IL-12 mRNA following measles virus infection of oligo-persistent JHMV infections. TNF-a mRNA is not translated in JHMV-infected cells (Stohlman et al., 1995a ), al-dendroglia (Yamabe et al., 1994 . During both the lethal and sublethal infections the IL-though it may be secreted by adjacent but not infected cells. In addition, inhibition of TNF-a, which prevents 1a mRNA peaks appear to coincide with replication and not clearance, suggesting that infection induces a rapid experimental autoimmune encephalitis (Ruddle et al., 1990) , has no effect on either JHMV-induced encephalitis induction of IL-1a mRNA. These data contrast to the association of IL-1a mRNA and the clearance of the OBLV-or demyelination (Stohlman et al., 1995a) . As anticipated, based on the relative tropism of the two viruses analyzed, 60 variant of JHMV (Pearce et al., 1994) , suggesting an additional difference in cytokine responses depending TNF-a mRNA accumulated initially in the CNS of mice infected with JHMV. However, by Day 5 p.i. there was on the tropism of the virus analyzed. IL-1b mRNA, previously detected in the CNS of JHMV-infected mice little difference in the levels of TNF-a mRNA in the two groups. Finally, the level of TNF-a mRNA decreased with (Pearce et al., 1994) , increased directly after infection at Day 1 p.i. However, the level was approximately the same suggests it may play a positive role in reducing the extent of CNS inflammation thereby inadvertently contributing as the level detected in sham-infected mice, suggesting it was induced by trauma. In all mice the levels subse-to persistent infection. Some aspects of our data, i.e., the rapid induction of IL-12 mRNA in mice infected with 2.2v-quently dropped by Day 3 p.i. The levels of IL-1b mRNA peaked at Day 5 following 2.2v-1 infection and at Day 7 1, suggest that infection of specific cell types may influence the induction of cytokine mRNA (Yamabe et al., following JHMV infection, suggesting IL-1b mRNA was also induced by infection. Analysis of the levels in immu-1994) . This supports the notion that the cytokine mRNA patterns more closely reflect diversity of the immune re-nodeficient mice were consistent with the notion that infection, and not immune infiltrates, contributed the ma-sponse to an individual agent, although differential secretion of cytokines following infection of unique CNS cell jority of the IL-1b mRNA levels.
IL-6, another pleiotropic cytokine with numerous ef-types cannot be ruled out (Benveniste, 1992; Sun et al., 1995) . While the kinetics of IFN-g, IL-4, and IL-10 showed fects on immune responses (van Snick, 1990) , was also detected early following both lethal and sublethal infec-little difference between the groups undergoing lethal or sublethal infections, mRNAs encoding IL-6 and IL-1b tions. By contrast, IL-6 mRNA was also only detected at 6 days p.i. with the neuronotropic OBLV-60 JHMV variant either appeared more rapidly (IL-6) or accumulated to higher levels (IL-1b) following infection with 2.2v-1 virus. (Pearce et al., 1994) . Kinetic analysis shows that the levels of IL-6 mRNA peaked at Day 3 post-2.2v-1 infection By contrast the induction of iNOS and IL-1a mRNAs were increased in mice undergoing a lethal infection. These and at Day 5 post-JHMV infection. Interestingly, analysis of the mRNA levels in the immunodeficient mice showed data suggest that an early induction of IL-6, and possibly IL-1b, are associated with sublethal infection or the dif-virtually no induction of IL-6 mRNA, suggesting that in contrast to IL-1a and IL-1b an intact immune response ferent tropisms exhibited by these two JHMV variants. However, during both infections the mRNA levels de-was required for IL-6 mRNA induction. Rapid induction of IL-6 mRNA following JHMV infection is consistent with creased as virus was cleared. Similarly, there appears to be an inverse correlation between a rapid induction other models of viral-induced encephalitis in which it also precedes IFN-g (Moskophidis et al., 1991) . Although of iNOS mRNA and sublethal disease, consistent with the recent demonstration that although iNOS is protective in both IL-6 and IL-10 are cofactors for CTL induction (Chen and Zlotnik, 1991; Takai et al., 1988) , kinetic analysis is vitro, inhibition of iNOS activity in vivo appears to have no effect on JHMV pathogenesis . Taken consistent with the notion that IL-6, and not IL-10, may be involved in the induction or recruitment of JHMV-specific together, kinetic analyses of the induction of cytokine mRNA during the lethal and sublethal JHMV infections CTL. JHMV infection induces IL-6 secretion from both brain endothelial cells and astrocytes following in vitro are consistent with the accumulation of both Th1-and Th2-associated cytokines and support the interaction of infection with JHMV (Joseph et al., 1993) , consistent with data showing that it is produced by resident CNS cells multiple cellular and soluble effector mechanisms whose balance may be critical in providing protection and steri-following infection with lymphocytic choriomeningitis virus (Frei et al., 1989) . It is interesting that IL-6 mRNA lizing immunity. peaks first in mice infected with the 2.2 v-1 variant compared to JHMV, which infects a significantly larger num-ACKNOWLEDGMENTS ber of astrocytes. The rapid induction of IL-6 and IL-We thank Wen-Qiang for excellent technical assistance. This work 1b following infection with 2.2v-1 is consistent with the was supported by Grant NS18146 from the National Institutes of Health.
induction of these mRNA in oligodendroglia infected by 