Experimental autoimmune encephalitis and inflammation in the absence of interleukin-12

Abstract

MethodsPeptide, antibodies, and ELISA.MOG 35-55 peptide (MEVGWYRSPFSRVVHLY-RNGK) was obtained from Research Genetics (Huntsville, Alabama, USA).ELISAs for IL-2, IL-4, IFN-γ, and IL-12 p70 were performed with kits purchased from Pharmingen (San Diego, California, USA), following the manufacturer's guidelines.All antibodies for flow cytometry (CD45, CD11b, CD8α, CD4, and GR-1) were preconjugated with either phycoerythrin, FITC, PerCP, or biotin, and were purchased from Pharmingen. Induction of EAE.Female C57BL/6 mice were obtained from NCI Laboratories (Frederick, Maryland, USA).Homozygous IL-12 p35 -/-and IL-12 p40 -/-C57BL/6 mice were originally purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and were bred in-house under pathogenfree conditions.Female C57BL/6, IL-12 p40 -/-, and p35 -/-mice 5-8 weeks old were immunized subcutaneously with 200 µg of MOG 35-55 peptide emulsified in CFA supplemented with 5 mg/ml of Mycobacterium tuberculosis (H37RA; Difco Laboratories, Detroit, Michigan, USA).The mice received intraperitoneal injections with 250 ng pertussis toxin (Sigma-Aldrich, St. Louis, Missouri, USA) at the time of immunization and