The Role of IL-10 in Mouse Hepatitis Virus-Induced Demyelinating Encephalomyelitis

Mark T. Lin,* David R. Hinton,*,†,1 Beatriz Parra,‡ Stephen A. Stohlman,†,‡ and Roel C. van der Veen†

*Department of Pathology, ‡Department of Molecular Microbiology and Immunology, and †Department of Neurology, School of Medicine,University of Southern California, Los Angeles, California 90033

Received September 22, 1997; returned to author for revision November 17, 1997; accepted March 31, 1998

Interleukin 10 (IL-10) is an important anti-inflammatory cytokine. To examine its role in virus-induced encephalomyelitis,IL-10-deficient (IL-10 2/2) mice were infected with a neurotropic strain of mouse hepatitis virus (JHMV). JHMV-infected IL-102/2 mice, compared to IL-4 2/2 and syngeneic C57BL/6 mice, exhibited increased morbidity and mortality. Virus wascleared from the CNS of all groups of mice with equal kinetics by day 9 postinfection and the lack of either IL-4 or IL-10 didnot alter the distribution of viral antigen, suggesting a lack of correlation between viral replication and the increased clinicaldisease in IL-10 2/2 mice. In moribund IL-10 2/2 mice, a moderate increase in mononuclear cell infiltration was correlatedwith increased expression of tumor necrosis factor-a, interferon-g, and inducible nitric oxide synthase mRNAs. In the smallpercentage of IL-10 2/2 mice that survived, no differences in either demyelination or inflammation were observed. Together,these results suggest that IL-10 is not required for viral clearance, and although it appears to be one of the mechanismsresponsible for inhibiting the extent of inflammation in the CNS during acute JHMV infection, it has little role in the eventualresolution of CNS inflammatory responses. © 1998 Academic Press

INTRODUCTION

Immunity to viral infections of the central nervoussystem (CNS) must attempt to balance the limitation ofviral replication and its cytopathology with the preserva-tion of CNS function. Infection of the CNS by the neuro-tropic JHM strain of mouse hepatitis virus (JHMV) pro-vides one example of these complex responses. JHMVinfection produces an acute panencephalomyelitiswhich progresses to persistent infection accompaniedby chronic ongoing demyelination (Weiner, 1973). Elimi-nation of infectious virus is important for protection fromlethal encephalitis; however, protection and viral clear-ance may be distinct immunological events (Kyuwa andStohlman, 1990; Houtman and Fleming, 1996; Lane andBuchmeier, 1997). Some immune effectors, such as cy-totoxic T lymphocytes (CTL), prevent death via directlyreducing CNS viral replication (Stohlman et al., 1995a;Lin et al., 1997), while other components, for instancevirus-specific CD41 T cells, may protect without alteringviral replication (Stohlman et al., 1986; Korner et al., 1991;Yamaguchi et al., 1991). Elimination of detectable infec-tious virus, without complete elimination of viral antigenor viral RNA (Perlman and Ries, 1987; Adami et al., 1995),suggests a moderated anti-viral immune response de-signed to preserve CNS function at the expense of viralelimination, thereby contributing to persistent infection.

Analysis of cytokine mRNA within the CNS during JHMVinfection showed that both IL-10 and IL-4 mRNA accu-mulate with kinetics similar to the mRNA encoding theproinflammatory cytokines, interferon-g (IFN-g) and tu-mor necrosis factor-a (TNF-a) (Parra et al., 1997). Whilethe induction of IL-10, TNF-a, and IFN-g mRNA levelswere similar, induction of IL-4 mRNA was considerablylower (Parra et al., 1997), suggesting that few T cellssecreting Th2-type cytokines are recruited into the CNSduring JHMV infection.

Several mechanisms for modulation of immune re-sponses within the CNS have been proposed. For exam-ple, lipids within the local CNS environment have beensuggested to limit viral-induced inflammation by inhibit-ing IL-2 production from infiltrating lymphocytes (Irani etal., 1997). Th2-derived cytokines, including IL-10, are im-portant in prevention of experimental allergic encepha-lomyelitis (EAE) (Rott et al., 1994) and correlate withdisease remission (Kennedy et al., 1992; Issazadeh et al.,1996). IL-10 suppresses macrophage functions as wellas Th1-type T cell responses (Moore et al., 1993). IL-4protects from neuronal injury via inhibition of TNF-a andnitric oxide (NO) production by activated microglia (Chaoet al., 1993). By contrast, the expression of TNF-a isassociated with progression of EAE (Navikas and Link,1996). Microglia express TNF-a during JHMV-inducedencephalomyelitis (Stohlman et al., 1995b; Sun et al.,1995); however, TNF-a appears to play no direct role inthe progression of disease (Stohlman et al., 1995b).These data suggested that at least with respect to TNF-a,JHMV-induced inflammation is distinct from the inflam-

1 To whom reprint requests should be addressed at University ofSouthern California School of Medicine, MCH 142, 1333 San Pablo St.,Los Angeles, CA 90033. Fax: 213-225-2369.

VIROLOGY 245, 270–280 (1998)ARTICLE NO. VY989170

0042-6822/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

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mation associated with CNS autoimmune disease. Inthis report, the potential role of IL-10 to modify viral-induced encephalomyelitis was examined by infection ofmice rendered genetically deficient in IL-10 via homolo-gous gene recombination.

RESULTS

To examine the potential role(s) of IL-10 in JHMV in-fection, clinical disease progression and mortality inIL-10 2/2 mice were compared to those of IL-4 2/2mice and syngeneic C57BL/6 mice. All groups of micedeveloped mild encephalitis (ruffled fur, hunched back,minor gait abnormality) between days 7 and 8 p.i., pro-gressed to moderate paraparesis by day 13, and showedsigns of recovery by day 18 p.i. (Fig. 1A). IL-10 2/2 miceexhibited a transient, yet significant, increase (P value ,0.05) in disease, compared to either the IL-4 2/2 orC57BL/6 control mice, between days 8 and 10 p.i. (Fig.1A). At the peak of clinical disease (day 11 p.i.), differ-ences were no longer statistically significant, althoughthe IL-10 2/2 group exhibited higher average clinicalgrades than the other groups throughout the remainderof the experiment (Fig. 1A). A reduced survival rate wasfound in the IL-10 2/2 group (Fig. 1B), consistent with

the increased clinical scores. Approximately one-third ofmice in both the IL-4 2/2 and C57BL/6 groups suc-cumbed to JHMV infection (Fig. 1B). By contrast, a muchhigher proportion (P value , 0.005) of IL-10 2/2 micedied between days 9 and 14 p.i. (Fig. 1B). Mean day ofsurvival for the IL-10 2/2 group was 12.8 days comparedto the IL-4 2/2 (19.1 days) and the control group (18.6days). These data suggest that IL-10, but not IL-4, isimportant for survival during JHM-induced encephalomy-elitis, consistent with the low level of IL-4 and higherlevel of IL-10 mRNA associated with JHMV infection(Parra et al., 1997).

IL-10 is associated with decreasing CNS inflammation(Rott et al., 1994; Navikas and Link, 1996) and autoim-mune CNS disease remission (Kennedy et al., 1992;Issazadeh et al., 1996) and is an important anti-inflam-matory cytokine limiting immune responses during avariety of systemic infections (Kuhn et al., 1993; Moore etal., 1993; Gallo et al., 1994; Tonnetti et al., 1995; Gazzinelliet al., 1996; Dai et al., 1997; Hunter et al., 1997). Todetermine whether the increased morbidity and mortalityobserved in JHMV-infected IL-10 2/2 mice correlatedwith increased or prolonged mononuclear cell infiltration,histopathological changes in the CNS of IL-10 2/2, IL-42/2, and C57BL/6 mice were compared. At day 7 p.i., allgroups had similar numbers and distribution of inflam-matory foci, consistent with the absence of a differencein clinical disease at this time point (data not shown).Only a moderate increase in CNS inflammation wasobserved in the IL-10 2/2 mice when compared to thecontrol groups at day 9 p.i. (Figs. 2A and 2B), consistentwith the transient increase in clinical scores observed(Fig. 1A). By day 18 p.i., the surviving IL-10 2/2 mice hadsimilar numbers of spinal cord demyelination plaques(IL-10 2/2 mice, 6.1 6 1.9, and control mice, 7.6 6 1.3)and cellular infiltrates when compared to control mice(Figs. 2C and 2D), suggesting that IL-10 does not influ-ence the development of JHMV-induced demyelination.Furthermore, no infectious virus was recovered fromthese survivors, although viral antigen, indicative of per-sistent infection, was still present (data not shown).These data suggest that even in the absence of IL-10, theimmune response remains unable to completely elimi-nate virus from the CNS.

JHMV clearance from the CNS is predominantly de-pendent on T cell immunity (Kyuwa and Stohlman, 1990;Stohlman et al., 1995a; Lane and Buchmeier, 1997). BothIL-10 and IL-4 are important T cell growth and maturationfactors (Widmer and Grabstein, 1987; Chen and Zlotnik,1991), in addition to their potential anti-inflammatoryroles (Moore et al., 1993; Powrie et al., 1993; Brown andHural, 1997). Therefore, JHMV replication in the CNS ofIL-10 2/2, IL-4 2/2, and C57BL/6 mice was comparedto test the possibility that the increased clinical scoresobserved in IL-10 2/2 mice reflected an inability toeliminate infectious virus. A small but significant in-

FIG. 1. Morbidity and mortality of IL-10 2/2, IL-4 2/2, and C57BL/6mice undergoing JHMV infection. (A) Groups of 16 to 20 IL-10 2/2, IL-42/2, and C57BL/6 were infected ic with 25 PFU JHMV and observedfor clinical scores as described under Materials and Methods. (*)Denotes statistical significance (P , 0.05, Kruskal–Wallis test). (B)Survival rates of JHMV-infected IL-10 2/2, IL-4 2/2, and C57BL/6 mice(P , 0.005, Mantel–Cox test).

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FIG. 2. Histology of CNS in JHMV-infected IL-10 2/2 and C57BL/6 mice. In (A), a brain section from an IL-10 2/2 mouse at day 9 p.i. showsmultifocal cellular infiltrates (arrows). In (B), a section from a similarly infected C57BL/6 mice shows less prominent inflammation (arrows). At 18 daysp.i., spinal cords of both IL-10 2/2 (C) and C57BL/6 (D) mice show demyelination (arrows) and mild chronic inflammation. Hematoxylin and eosinstain. Magnification, 3110.

272 LIN ET AL.

crease in infectious virus was detected in both the IL-102/2 and IL-4 2/2 mice compared to C57BL/6 mice atday 7 p.i. (Fig. 3A). However, infectious virus was clearedfrom the CNS of all groups by day 9 p.i. (Fig. 3A). To

confirm the transient increase in virus detected at day7 p.i. and complete virus clearance by day 9 p.i., repli-cation was also examined in infected C57BL/6 micedeleted of IL-4 or IL-10 via antibody treatment. Antibody-

FIG. 2—Continued

273ROLES OF IL-10 IN JHMV CNS INFECTION

mediated IL-4 and IL-10 depletions also resulted in atransient increase in virus replication at day 7 p.i.(Fig.3B). Similarly, no infectious virus was detected ineither antibody-treated group at day 9 p.i.. These dataconfirm that both IL-4 and IL-10 contribute to the earlyclearance of virus from the CNS; however, neither arenecessary for complete elimination of infectious virus. Inaddition, although more CD41 and CD81 T cells werepresent in the CNS of the IL-10 2/2 mice compared tocontrol mice at day 9 p.i. (data not shown), the ratio ofCD41 to CD81 lymphocytes remained at 2:1 in bothgroups, suggesting that IL-10 does not participate in thedifferential regulation of T cell trafficking into the CNS.Infectious virus could not be detected at the time when

most of the IL-10 2/2 mice succumb to infection (day9-14 p.i., Fig. 1B). These data suggest that the increasedmorbidity and mortality observed in IL-10 2/2 mice wasnot due to changes in JHMV replication.

Distribution of viral antigen was examined to deter-mine if the tropism of JHMV was altered during infectionof the IL-10 2/2 mice and had thus contributed to theincreased morbidity and mortality. No difference in thenumbers of antigen-positive cells was observed compar-ing all groups at day 7 p.i. (data not shown); however,increased numbers of antigen-positive cells were ob-served in the IL-10 2/2 mice at day 9 p.i. (Figs. 4A and4B) when infectious virus was no longer detectable.Importantly, at no point during the infection was a differ-ence in JHMV-infected cell types noted, suggesting thatthe absence of IL-10 did not contribute to clinical diseaseby altering CNS cellular tropism. Although IL-10 2/2mice succumb to chronic enterocolitis (Kuhn et al., 1993),no evidence of inflammatory disease or secondary infec-tion was detected in colon, kidney, and liver from ran-domly selected moribund IL-10 2/2 mice (data notshown). Systemic infections of IL-10 2/2 mice are alsoassociated with increased TNF-a expression correlatingwith tissue destruction and morbidity (Gazzinelli et al.,1996; Dai et al., 1997). To determine if increased TNF-aexpression correlated with the morbidity and mortality ofthe JHMV-infected IL-10 2/2 mice, TNF-a mRNA expres-sion within the CNS and serum levels of TNF-a wereexamined. In the CNS of moribund IL-10 2/2 mice at day9 p.i., a threefold increase in TNF-a mRNA level wasdetected over that of the control mice (Fig. 5); however,no serum TNF-a was detected (, 2.5 units/ml) (data notshown), confirming the absence of a systemic inflamma-tory process. In addition to CNS TNF-a mRNA expres-sion, the levels of IFN-g and iNOS, both indicators ofinflammatory responses, and TGF-b mRNA, an anti-in-flammatory cytokine, were also examined (Fig. 5). Theamount of CNS IFN-g and iNOS mRNA expression wasalso increased three- to fourfold in moribund IL-10 2/2mice. By contrast, the amount of TGF-b mRNA was notdifferent between the two groups. The amounts of TNF-a,iNOS, IFN-g, and TGF-b mRNA expression in theC57BL/6, IL-4 2/2, and nonmoribund IL-10 2/2 micewere similar (data not shown), indicating that increasedviral burden did not contribute to the increased expres-sion of proinflammatory cytokines in the IL-10 2/2 mice.These data suggest that increased expression of inflam-matory cytokines due to a lack of IL-10 regulation couldcontribute to the increased morbidity and mortality in theIL-10 2/2 mice.

DISCUSSION

Infection of the CNS by JHMV results in a vigorousimmune infiltrate, composed of natural killer cells, CD41

T cells, and CD81 T cells (Dorries et al., 1991; Williamson

FIG. 3. JHMV titer in the brain of JHMV-infected mice. (A) Virus titersin IL-10 2/2, IL-4 2/2, and control mice and (B) anti-cytokine mAb-treated mice at days 7 and 9 p.i. To deplete IL-4 and IL-10, C57BL/6mice were injected intraperitoneally with 1 mg of anti-IL-4 (11B11) oranti-IL-10 (JES5-2A5) mAbs at 2 days prior to and 2 days post JHMVinfection. Virus titers were determined by plaque assay as describedunder Materials and Methods. Each time point represents the mean offour samples and the standard errors are expressed as error bars.Student’s t test was used to test for significance. (*) Denotes P , 0.05and (–) denotes titers below detection level (,200 PFU).

274 LIN ET AL.

FIG. 4. JHMV antigen in the CNS of IL-10 2/2 and C57BL/6 mice. In (A), a brain section from an IL-10 2/2 mouse at day 9 p.i. shows increasednumbers of antigen-positive cells (arrows) compared to similarly infected C57BL/6 mice (B). In each group, a mixed population of glial cells and smallnumbers of neurons are stained. Immunoperoxidase stain using the ABC method for viral antigen in conjunction with anti-JHMV mAb J.3.3 andcounterstained with hematoxylin and eosin. Magnification, 3440.

275ROLES OF IL-10 IN JHMV CNS INFECTION

et al., 1991; Williamson, 1992). The intent of this immuneresponse is to prevent lethal viral-induced encephalitis(Kyuwa and Stohlman, 1990). However, these effectorsare unable to provide a sterilizing immunity within theCNS, resulting in persistent viral antigen and RNA (Perl-man and Ries, 1987; Adami et al., 1995), associated withongoing primary demyelination (Weiner, 1973). Thus, itappears that the anti-viral effector mechanisms withinthe CNS are suppressed prior to the complete elimina-tion of virus. Consistent with these findings, the confinedspace of the cranial cavity combined with the inability ofneurons to regenerate necessitates tightly controlled im-mune responses within the CNS. In many viral infections,functionally polarized responses as measured by anti-viral antibody response or Th1-type cytokines are impor-tant for viral clearance during acute infection (Kurilla etal., 1993; Finke et al., 1995; Moran et al., 1996; Willenborget al., 1996). Analysis of cytokine mRNA induction duringJHMV-induced encephalitis showed that IL-10 mRNA ac-cumulates with kinetics corresponding to the accumula-tion of IFN-g mRNA. By contrast, the magnitude of theIL-4 mRNA accumulation in the CNS was dramaticallyless than either IL-10 or IFN-g during both lethal andsublethal JHMV infections. These data were interpretedto suggest that few Th-2-type T cells infiltrate the CNSduring JHMV infection (Parra et al., 1997). This interpre-tation is consistent with both the data in this reportdemonstrating no alterations in the pathogenesis in IL-4

2/2 mice and recent analysis of JHMV pathogenesis inmice deficient in IFN-g, in which an increase in IL-4mRNA was found in the peripheral organs, but not theCNS (unpublished data). Together, these data suggestthat the increased IL-10 mRNA occurs predominantly inCNS resident cells, in contrast to the active recruitmentof Th2-type T cells into the inflamed CNS. In support ofthis view, it had been shown recently that normal micro-glia/macrophages express IL-10, and in response to Toxo-plasma infection, these cells had increased expressionof IL-10 (Schluter et al., 1997). In our model, preliminaryimmunostaining of JHMV-infected C57BL/6 brain sec-tions also showed positive expression in cells morpho-logically identified as perivascular mononuclear cellsand parenchymal microglia (data not shown).

IL-10 is associated with both the prevention of EAE(Rott et al., 1994) and with the remission phase of EAE(Kennedy et al., 1992; Issazadeh et al., 1996) which fol-lows an active attack of CNS cells by self-reactive Th1-type T cells. The present study examined the propositionthat IL-10, predominantly secreted within the local CNSenvironment, might contribute to limiting immune effec-tors prior to complete clearance of infectious virus. Con-sistent with this notion, CNS infection with JHMV re-sulted in increased morbidity and mortality in IL-10 2/2mice. However, comparisons with the pathogenesis insyngeneic control and IL-4 2/2 mice showed that theincreased morbidity and mortality was not directly attrib-utable to a large increase in inflammation, a delay in viralclearance, or altered cellular tropism. These data agreewith the observed increased mortality of IL-10 2/2 miceinfected with either Toxoplasma gondii (Gazzinelli et al.,1996) or Trypanosoma cruzi (Hunter et al., 1997), neitherof which are associated with increased parasite load.Similarly, histopathological changes in disease modelsexamining the effects of IL-10 deficiency have been vari-able. Infection of the IL-10-deficient mice with Listeriamonocytogenes or T. cruzi results in reduced tissue de-struction, even in the presence of strong Th1-type re-sponses (Dai et al., 1997; Hunter et al., 1997). By contrast,in both contact hypersensitivity (Berg et al., 1995) andsystemic T. gondii infections (Gazzinelli et al., 1996), theabsence of IL-10 results in severe tissue necrosis. Thedata presented in this report indicate that neither IL-10nor IL-4 appears to be required for the suppression ofJHMV-induced CNS inflammation. These results contrastwith the proposed anti-inflammatory roles of IL-10 andIL-4 during autoimmune-mediated CNS inflammation(Rott et al., 1994; Kennedy et al., 1992; Racke et al., 1995;Issazadeh et al., 1996; Shaw et al., 1997) and suggestthat these effects may reflect influences on the regula-tion of autoimmune effector cells. These results furthersuggest that other mechanisms of immune regulationmay have greater roles in suppressing inflammation inthe CNS. Growth factor deprivation has been suggestedas a potential mechanism of immune suppression within

FIG. 5. Expression of TNF-a , IFN-g, iNOS, and TGF-b mRNA in thespinal cord of IL-10 2/2 and control mice at day 9 p.i. SemiquantitativeRT–PCR analyses were performed as described under Materials andMethods. For each cytokine analysis, samples with the highest mRNAexpression were designated as 100% maximum response and theremaining data were expressed as a percentage of the maximum. Datashown are the mean of three mice 6 1 standard deviation.

276 LIN ET AL.

the CNS during viral-induced encephalitis (Welsh et al.,1995). In addition, TGF-b, another potent downregulatorof inflammation, has also been suggested to function inthe CNS (Biron, 1994; Navikas and Link, 1996). Although,the role of TGF-b in JHMV pathogenesis remains unclear,IL-10 2/2 mice express amounts of TGF-b mRNA ap-proximately equal to that of the control group, suggestinga potential downregulatory role for TGF-b in JHMV CNSinfection.

Increased mortality in IL-10 2/2 mice correlated withonly a modest increase in CNS inflammation and a mod-erate increase in the expression of inflammatory cyto-kines. TNF-a selectively damages myelin, is cytotoxic tooligodendrocytes (Selmaj and Raine, 1988), and is asso-ciated with edema due to alterations in blood–brain bar-rier permeability (Mustafa et al., 1989). However, duringJHMV infection, TNF-a depletion does not alter the mor-bidity, mortality, extent of encephalitis, or demyelination(Stohlman et al., 1995b). In addition to its direct cyto-pathic effects, both TNF-a and IFN-g are potent inducersof NO production, which has been correlated with theseverity of neuropathology in various viral infections inthe CNS (Campbell et al., 1994; Hooper et al., 1995;Tucker et al., 1996). As with TNF-a, although the detec-tion of elevated IFN-g and iNOS messages (Grzybicki etal., 1997; Parra et al., 1997) might suggest that NO couldcontribute to the increased morbidity and mortality, re-cent data have demonstrated that JHMV replication andJHMV-induced pathology are not altered in mice treatedwith iNOS inhibitor (Lane et al., 1997). These data sug-gest that TNF-a and iNOS by themselves may not directlycontribute to the pathogenesis of JHMV-induced enceph-alomyelitis; however, it still remains possible that theycould contribute concurrently to the disease process.Results from this report suggest that IL-10 may suppressthe expression of these inflammatory cytokines in theCNS. This lack of suppression may be the factor explain-ing the decreased survival of IL-10 2/2 mice at thecritical time point where even normal mice succumb toJHMV infection. These data support a role for IL-10 inbalancing the preservation of CNS functions with viralelimination during JHMV infection.

The observations on the lack of an important role forTNF-a (Stohlman et al., 1995b), iNOS (Lane et al., 1997),and IL-4 (Parra et al., 1997) during viral-induced enceph-alomyelitis, compared to their effects on autoimmune-mediated CNS inflammation (Selmaj and Raine, 1988;Ruddle et al., 1990; Navikas and Link, 1996), suggest thatthese two processes are regulated via independentmechanisms. In support of this notion, the differentialregulation of two Th1-mediated inflammatory responses,contact sensitivity (Berg et al., 1995) and delayed-typehypersensitivity (Powrie et al., 1993), by IL-10 suggeststhat viral-induced encephalomyelitis is more similar tothe mechanisms associated with delayed-type hypersen-

sitivity, while EAE is more similar to the mechanismsregulating contact sensitivity.

MATERIALS AND METHODS

Experimental animals

Seven- to eight-week-old male (H-2b) IL-10 2/2 andIL-4 2/2 mice were kindly provided by Dr. R. Coffman(DNAX Research Institute, Palo Alto, CA). The homozy-gosity of the deletions was confirmed by PCR genotyp-ing. Age- and sex-matched C57BL/6 mice were pur-chased from the Jackson Laboratories (Bar Harbor, ME).All mice were seronegative for MHV antibody and weremaintained under specific pathogen-free conditions.

Virus and titer determination

Mice were infected by intracerebral (ic) inoculationwith 25 PFU of the 2.2v-1 monoclonal antibody (mAb)escape variant of JHMV contained in 32 ml of phosphate-buffered saline (PBS). The 2.2v-1 virus, kindly supplied byDr. J. Fleming (University of Wisconsin), produces a sub-lethal infection with severe demyelinating disease (Flem-ing et al., 1986) and was tested for reversion prior to use.JHMV replication in the CNS was determined by plaqueassay on monolayers of DBT cells as previously de-scribed (Stohlman et al., 1986). Briefly, one half of thebrains was homogenized in 2.0 ml of Dulbecco’s PBS (pH7.4), using Ten Broeck tissue homogenizers. The otherhalf was processed for histology (see below). Followingcentrifugation at 200 g for 7 min at 4°, supernatants wereeither assayed immediately or frozen at 270°C. Datapresented are the average of triplicate determinations forgroups of four or more mice.

Clinical scores

Clinical disease was graded as previously described(Fleming et al., 1988): 0, healthy; 1, ruffled fur andhunched backs; 2, slow mobility and inability to return toupright position when turned on its back; 3, paralysis andwasting; 4, moribund and death. Average clinical scoresfor at least four mice per group are reported.

Cytokine mRNA expression

RNA was prepared from half spinal cords by homog-enization in guanidine isothiocyanate and isolated bycentrifugation through 5.4 M cesium chloride at 100,000g for 18 h as previously described (Cua et al., 1995; Parraet al., 1997). The cDNA were prepared using avian my-eloblastosis reverse transcriptase (Promega, Madison,WI) and oligo(dT) primers (Promega) for 60 min at 42°C.Expression of cytokine mRNA was determined by semi-quantitative PCR analysis, following procedures de-scribed previously (Cua et al., 1995; Parra et al., 1997).PCR was performed using AmpliTaq DNA polymerase(Perkin–Elmer, Branchburg, NJ) and specific cytokine

277ROLES OF IL-10 IN JHMV CNS INFECTION

primers for TNF-a, IFN-g, and iNOS as previously de-scribed (Parra et al., 1997). For quantification, PCR prod-ucts were diluted in denaturing solution (0.4 N NaOH, 25mM EDTA), neutralized with Tris–HCL (1.0 M; pH 8.0), andtransferred to 0.45-mm Nytran membranes (Schleicher &Schuell, Keene, NH) using a Minifold I dot blot apparatus.Membranes were hybridized (60°C, overnight) with[32P]ATP-labeled internal oligonucleotide probes. Mem-branes were washed (three times; 23 SSC, 0.1% SDS;room temperature), exposed to Storage phosphorScreens (Molecular Dynamics, Sunnyvale, CA), andscanned using phosphorimaging scanner (Molecular Dy-namics). To adjust for differences in the amount of cDNA,cytokine cDNA levels were normalized to the house-keeping enzyme hypoxanthine phosphoriboxyltrans-ferase (HPRT) as previously described (Cua et al., 1995;Parra et al., 1997). For each cytokine, the highest meanspecific activity was designated as 100% maximal re-sponse. The remainder of the samples were expressedas a percentage of the highest value. Data shown aremean value of three mice 6 1 standard deviation.

In vivo depletion of cytokines

Groups of C57BL/6 mice were treated intraperitoneallywith 1 mg of purified anti-IL-4 (11B11), or anti-IL-10 (JES5-2A5), or an isotype-matched control anti-b-galactosidasemAb (GL113) (kindly provided by Dr. R. Coffman, DNAXResearch Institute, Palo Alto, CA) 2 days prior to and 2days postinfection (p.i.). We had shown previously thatrat mAb of the same isotype when similarly administeredwas able to cross the blood–brain barrier during JHMVinfection (Stohlman et al., 1995b). Purified mAbs wereprepared from serum-free culture supernatants and con-tained less than 3 EU of endotoxin/mg.

Histology

One half of the brains and the spinal cords from threeto four mice per group were fixed for 3 h in Clark’ssolution (75% ethanol, 25% glacial acetic acid) and em-bedded in paraffin. Distribution of JHMV antigen wasexamined by immunoperoxidase staining (Vectastain-ABC kit; Vector Laboratory, Burlingame, CA) using theanti-JHMV mAb J.3.3 specific for the carboxy terminus ofthe N protein as primary antibody (Fleming et al., 1983)and horse anti-mouse monoclonal antibody as the sec-ondary antibody (Vector Laboratory, CA) and then coun-terstained with hematoxylin and eosin. Multiple serial-step paraffin sections of the spinal cord were stainedwith luxol fast blue. The number of demyelinatingplaques was counted to compare the extent and distri-bution of demyelinating lesions. CD41 and CD81 T cellswere identified by immunostaining acetone-fixed frozensections using anti-CD4 (H129.19, Pharmingen, San Di-ego, CA) and anti-CD8 (53-6.7, Pharmingen) antibodies at1:200 dilution. Primary antibodies were then detected

with biotin-conjugated rabbit anti-rat antibody preab-sorbed with mouse serum (Vector Laboratory) and Vec-stain-ABC kit.

Statistics

For comparison of clinical scores and mortality, theKruskal–Wallis and the Mantel–Cox tests were per-formed, respectively. Differences in virus titers and thenumbers of demyelinating plaques were compared usingthe Student’s t test.

ACKNOWLEDGMENTS

We thank Wen-Qiang and Ligaya Pen for technical assistance andLaurie Lebree for help in statistical analysis. This work was supportedby Grants NS18146 and EY03040 from the National Institute of Health.

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