2 cells, using protein G columns according to standard protocols. Soluble TNFR1 fusion protein (sTNFR1-Ig) was a kind gift from Geoff Hale (Therapeutic Antibody Group, University of Oxford, UK). All fluorochrome-conjugated anti-mouse mAbs and secondary detection reagents used were purchased from BD Biosciences (Oxford, UK). Biotinylated anti-CD3ζ was from Upstate (Watford, UK), and purified polyclonal rabbit anti-mouse EP1, EP2, EP3 and EP4 were from Cayman Chemicals (Ann Arbor, MI). Bone marrow (BM) Mϕ were generated using a method adapted from Munder et al.21 Briefly, bone marrow cells were resuspended at 5 × 105
cells/ml see more in complete media supplemented with 5% v/v horse serum (Invitrogen), and 50 pg/ml macrophage colony-stimulating factor. The cell suspension was transferred to hydrophobic PTFE-coated tissue culture
bags (supplied by Dr M. Munder, University of Heidelberg, Heidelberg, Germany) and incubated for 8 days at 37° in 5% v/v CO2. Single-cell splenocyte suspensions were generated by grinding spleens through a 70-μm cell strainer (BD Biosciences) with a syringe plunger. When used as APCs, splenocytes were irradiated with 3000 Rads using a caesuim-137 source (Gravatom, Hants, UK). The OT-II CD4+ T cells were prepared by enriching CD4+ cells from single cell suspensions of C57BL/6 OT-II splenocytes, using anti-CD4 microbeads (Miltenyi Biotech, Bisley, UK) according to the manufacturer’s instructions. B cells were prepared from spleens using anti-B220 microbeads (Miltenyi Biotech). Doxorubicin chemical structure Dendritic cells were generated from cultures of bone marrow cells as previously described.22 The 1 × 105 APCs were co-cultured with CD4+ T cells at ratio of 1 : 1 in round-bottom 96-well plates in complete media. The OVA peptide was added at the indicated concentrations. To some cultures the arginine analogue, l-NG-monomethyl arginine, the NO donor S-nitroso-N-acetyl-l,l-penicillamine,
or the cyclo-oxygenase (COX) inhibitor indomethacin (all from Sigma) was added. In some experiments, recombinant IFN-γ (Peprotech, London, UK), or PGE2 (Sigma) was added. Cells were cultured in a humidified Amoxicillin environment at 37°, 5% v/v CO2. Proliferation was measured by pulsing with 18·5 kBq [3H]thymidine (GE Healthcare, Bucks, UK) per well for the final 8 hr of culture and determining thymidine uptake [measured in counts per minute (c.p.m.)]. Accumulated NO production was measured after 64 hr in culture supernatants using Griess reagent (Sigma) as previously described.23 Production of IFN-γ was assessed using a murine T helper type 1 (Th1)/Th2 Flow cytomix 10plex kit (Bender Medsystems, Vienna, Austria) according to the manufacturer’s instructions. Concentration of PGE2 was measured using an enzyme immunoassay competition enzyme-linked immunosorbent assay kit (Caymen Chemical, Ann Arbor, MI) according to the manufacturer’s instructions.