Interleukin-4 and interleukin-10 modulate nuclear factor jB activity and nitric oxide synthase-2 expression in Theiler’s virus-infected brain astrocytes Eduardo Molina-Holgado,* Angel Are ´valo-Martı ´n,* Antonio Castrillo, Lisardo Bosca ´,  Jose ´M.Velaà and Carmen Guaza* *Department of Neural Plasticity, Instituto Cajal, Consejo Superior de Investigaciones Cientı ´ficas, Madrid, Spain  Instituto de Bioquı ´mica (Centro Mixto Consejo Superior de Investigaciones Cientı ´ficas–UCM), Universidad Complutense, Madrid, Spain àDepartment of Cellular Biology and Physiology, Unitat d’Histologia, Universitat Auto ´noma de Barcelona, Bellaterra, Barcelona, Spain Abstract In brain astrocytes, nuclear factor jB (NF-jB) is activated by stimuli that produce cellular stress causing the expression of genes involved in defence, including the inducible nitric oxide synthase (NOS-2). Theiler’s murine encephalomyelitis virus (TMEV) induces a persistent CNS infection and chronic immune-mediated demyelination, similar to human multiple sclerosis. The cytokines interleukin (IL)-4 and IL-10 inhibit the expression of proinflammatory cytokines, counteracting the inflammatory process. Our study reports that infection of cul- tured astrocytes with TMEV resulted in a time-dependent phosphorylation of IjBa, degradation of IjBa and IjBb, acti- vation of NF-jB and expression of NOS-2. The proteasome inhibitor MG-132 blocked TMEV-induced nitrite accumulation, NOS-2 mRNA expression and phospho-IjBa degradation, suggesting NF-jB-dependent NOS-2 expression. Pretreat- ment of astrocytes with IL-4 or IL-10 decreased p65 nuclear translocation, NF-jB binding activity and NOS-2 transcription. IL-4 and IL-10 caused an accumulation of IjBa in TMEV- infected astrocytes without affecting IjBb levels. The IjB kinase activity and the degradation rate of both IjBs were not modified by either cytokine, suggesting de novo synthesis of IjBa. Indeed, IL-4 or IL-10 up-regulated IjBa mRNA levels after TMEV infection. Therefore, the accumulation of IjBa might impair the translocation of the NF-jB to the nucleus, mediating the inhibition of NF-jB activity. Overall, these data suggest a novel mechanism of action of IL-4 and IL-10, which abrogates NOS-2 expression in viral-infected glial cells. Keywords: glial cells, IjBa, NOS-2, TMEV, viral infection. J. Neurochem. (2002) 81, 1242–1252. Theiler’s murine encephalomyelitis virus (TMEV), a posit- ive-sense single-stranded RNA virus, is widely used as a model for human diseases such as multiple sclerosis (MS), poliomyelitis and postpolio syndrome (Theiler 1934; Lipton and Dal Canto 1976; Miller et al. 1997). The mouse is the natural host for this picornavirus and intracerebral injection of TMEV produces a neurological biphasic disease in susceptible SJL/J mice. Following the initial acute enceph- alitis,infectedmicedevelopachronicprimaryinflammatory axonal demyelination, with pathological abnormalities and neurologicaldeficitssimilartothoseobservedinMS.Atthis phaseofinfectionastrocytesandmicroglia/macrophages,the most abundant brain glial cells, are critical for virus persistencewithintheCNSandtocontrolbloodbrainbarrier permeability (Rodrı ´guez et al.1996;Zheng et al.2001). Resubmitted manuscript received March 4, 2002; accepted March 4, 2002. Address correspondence and reprint requests to Carmen Guaza, Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain. E-mail: cgjb@cajal.csic.es Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; ECL, enhanced chemiluminescence reagents; EMSA, electrophoretic mobility shift assay; FCS, fetal calf serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEPES, 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid; IFN, interferon; IRF-1, IFNregulatoryfactor-1;IjB,NF-jB inhibitory protein; IL, interleukin; IKK, IjB kinase; MS, multiple sclerosis; NF-jB, nuclear factor jB; NOS-2,induciblenitricoxidesynthase;PBS,phosphate-bufferedsaline; PMSF, phenylmethylsulfonyl fluoride; RT, room temperature; SDS, sodium dodecyl sulfate; STAT, signal transducers and activators of transcription; TBS, Tris-buffered saline; TMEV, Theiler’s murine encephalomyelitis virus; TNF-a, tumour necrosis factor a. Journal of Neurochemistry ,2002, 81, 1242–1252 1242 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 81, 1242–1252