The Inflammatory Response to Nonfatal Sindbis Virus Infection of the Nervous System Is More Severe in SJL Than in BALB/c Mice and Is Associated with Low Levels of IL-4 mRNA and High Levels of IL-10-Producing CD41 T Cells1 Jennifer F. Rowell and Diane E. Griffin2 SJL mice are susceptible to inflammatory autoimmune diseases of the central nervous system (CNS), while BALB/c mice are relatively resistant. To understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of SJL and BALB/c mice to infection with Sindbis virus, a virus that causes acute nonfatal encephalo- myelitis in both strains of mice. Clearance of virus was similar, but SJL mice developed a more intense inflammatory response in the brain and spinal cord and inflammation persisted for several weeks. Analysis of lymphocytes isolated from brains early after infection showed an absence of NK cells in SJL mice, while both strains of mice showed CD41 and CD81 T cells. During the second week after infection, CD41 T cells increased in SJL mice and the proportion of CD81 T cells decreased, while the opposite pattern was seen in BALB/c mice. Expression of IL-10 mRNA was higher and IL-4 mRNA was lower in the brains of infected SJL than in BALB/c mice, while expression of the mRNAs of IL-6, IL-1b, TNFa, and the Th1 cytokines IL-2, IL-12, and IFN-g was similar. Lymphocytes isolated from the CNS of SJL mice produced large amounts of IL-10. CNS lymphocytes from both strains of mice produced IFN-g in response to stimulation with Sindbis virus, but not in response to myelin basic protein. These data suggest that IL-10-producing CD41 T cells are differentially recruited to or regulated within the CNS of SJL mice compared with BALB/c mice infected with Sindbis virus, a characteristic that may be related to low levels of IL-4, and is likely to be involved in susceptibility of SJL mice to CNS inflammatory diseases. The Journal of Immunology, 1999, 162: 1624–1632. he AR339 strain of Sindbis virus (SV)3 is an alphavirus tion is first detectable 3–4 days after infection when mononuclear that causes acute, nonfatal, and usually asymptomatic en- cells start to cross the cerebrovascular endothelium to form T cephalomyelitis in weanling mice. After intracerebral in- perivascular cuffs. The inflammatory response is maximal after oculation, the virus replicates rapidly in the brain and spinal cord, 7–10 days and is usually resolved within 2–3 wk (2). primarily in neurons. A systemic and local immune response is Inflammation in the central nervous system (CNS) is rigorously by guest on September 30, 2021. Copyright 1999 Pageant Media Ltd. induced and virus replication is soon controlled with levels of in- controlled by several known mechanisms and probably additional fectious virus becoming undetectable within 7–8 days after infec- mechanisms yet to be identified. The blood brain barrier is a phys- tion. Elimination of infectious virus correlates with the appearance ical barrier that prevents entry of most immune cells. Activated T of neutralizing Ab and the development of a virus-specific mono- cells can pass through the blood brain barrier, but quickly exit the nuclear inflammatory response in the brain and spinal cord (1, 2). CNS in the absence of Ag recognition (3, 4). T cell stimulation by Adoptive transfer experiments using persistently infected immu- Ag present in the CNS is further inhibited due to low levels of nodeficient SCID mice have shown that Ab specific for the E2 MHC molecules, costimulatory molecules, and adhesion mole- glycoprotein of SV mediates clearance of infectious virus, even in cules, although cellular damage and inflammatory mediators can the absence of SV-specific cell-mediated immunity (1), so the role https://www.jimmunol.org increase expression (5, 6). of the inflammatory response in recovery is not clear. Inflamma- SV infection results in varying levels of CNS inflammation de- pending on the strain of mouse infected. Compared with BALB/c mice, SJL mice display more extensive inflammation and this in- Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205 flammation may persist for several weeks to months (7). SJL mice Received for publication June 17, 1998. Accepted for publication October 26, 1998. are also susceptible to several inflammatory autoimmune diseases Downloaded from The costs of publication of this article were defrayed in part by the payment of page of the CNS such as experimental autoimmune encephalitis (EAE) charges. This article must therefore be hereby marked advertisement in accordance (8, 9) and Theiler’s murine encephalomyelitis virus (TMEV)-in- with 18 U.S.C. Section 1734 solely to indicate this fact. duced demyelination (10), while BALB/c and many other strains 1 This work was supported by Research Grant NS29234, a Javits Neuroscience In- vestigator Award from the National Institutes of Health (D.E.G.), and by a postdoc- of mice are relatively resistant to these diseases. Both of these toral fellowship from the National Multiple Sclerosis Society (J.F.R.). murine demyelinating diseases serve as models for the human de- 2 Address correspondence and reprint requests to Dr. Diane E. Griffin, Department of myelinating disease multiple sclerosis, and more complete under- Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, standing of the inflammatory process may enhance our general 615 N. Wolfe Street, Baltimore, MD 21205. E-mail address: dgriffi[email protected] understanding of autoimmune diseases of the CNS. 3 Abbreviations used in this paper: SV, Sindbis virus; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; GAPDH, glyceraldehyde phos- EAE is induced by immunization with whole spinal cord or phate dehydrogenase; IP, IFN-g-inducible protein; MBP, myelin basic protein; MIP, components of myelin such as myelin basic protein (MBP) or pro- macrophage inflammatory protein; PE, phycoerythrin; PFU, plaque-forming units; PI, postinfection; TMEV, Theiler’s murine encephalomyelitis virus; TUNEL, terminal teolipid protein together with CFA (8, 11). Paralysis is accompa- deoxynucleotidyltransferase-mediated UTP nicked end labeling. nied by inflammation consisting of T cells, macrophages and B Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 1625 Table I. Abs used for analysis of inflammatory cells and for ELISA Determinant Recognizeda Clone Conjugate Source Method Macrophage/Monocyte F480 None Hybridoma supernatant IHCb CD45R/B220 RA3-6B2 None, FITC PharMingen, San Diego, CA IHC, FCc CD4 (L3T4) RM4-5 None, FITC PharMingen IHC, FC CD8a (Ly-2) 53-6.7 None, FITC PharMingen IHC, FC CD3e 145-2C11 PE PharMingen FC NK cells DX5 FITC PharMingen FC ab T cell receptor H57-597 FITC PharMingen FC gd T cell receptor GL3 FITC PharMingen FC IgM R6-60.2 FITC PharMingen FC Ig polyclonal HRPd Dako, Carpinteria, CA ELISA IgG1 G1-6.5 HRP PharMingen ELISA IgG2a R19-15 HRP PharMingen ELISA IL-10 (capture) JES5-2A5 None PharMingen ELISA IL-10 (detection) SXC-1 Biotin PharMingen ELISA a All Abs are specific for mouse determinants. b IHC, immunohistochemistry. c FC, flow cytometry. d HRP, horseradish peroxidase. 1 cells. The disease is mediated by CD4 Th1-type T cells that se- erate to large inflammatory foci in .50% of 310 fields. Scores were in- crete inflammatory cytokines such as IL-2 and IFN-g (12). The creased by up to 1 point with abundant parenchymal cellularity. inflammation persists for several days, then resolves. Cryopreserved sections were cut from OCT-embedded tissue and used for immunohistochemistry. Sections were blocked with 0.5% dry milk in TMEV infects neurons and oligodendroglia of the brain and PBS, and endogenous peroxidase activity was quenched with either 1% spinal cord. Resistant strains of mice clear virus during the acute H2O2 in absolute methanol or 0.3% H2O2 in 0.1% sodium azide. Sections phase of the infection. In SJL mice, virus clearance is incomplete were incubated overnight at 4°C, with primary Ab diluted in PBS contain- resulting in persistent infection of macrophages and glial cells. In ing 0.5% dry milk, washed, and incubated with biotinylated secondary Ab 1 (Vector Laboratories, Burlingame, CA) in PBS/0.5% dry milk. The signal these mice, TMEV-specific CD4 Th1-type T cells mediate a was amplified using the Vectastain Elite ABC kit (Vector) and detected chronic inflammatory process that results in demyelination and with 3,39-diaminobenzidine (Sigma, St. Louis, MO). For detection of CD8, paralysis (13, 14). the signal was further amplified using TSA-Indirect Tyramide Signal Am- The differences in the development and regulation of immune plification (DuPont NEN, Boston, MA). The primary Abs used in this study responses within the CNS of SJL mice compared with BALB/c are listed in Table I. Sections were examined under code for the level of inflammation, the proportion of positively staining cells in inflammatory mice that increase susceptibility to inflammatory diseases involve lesions, and the relative number of positively staining cells in the paren- genes primarily outside the H-2 region and are only partially de- chyma. Immunohistochemical staining was scored on coded slides using a by guest on September 30, 2021. Copyright 1999 Pageant Media Ltd. fined (10). To gain a better understanding of these differences, we scale of 0–3 in which 0 indicates no positive cells, 1 indicates 5–20%, 2 have examined the CNS inflammatory response in SJL and indicates 20–60%, and 3 indicates 60–100% positive inflammatory cells. Scores were adjusted by up to 1 point when the numbers of positively BALB/c mice during acute encephalitis induced by infection with staining cells in the parenchyma were considerably higher or lower than SV, a virus that is not associated with late neurologic disease.