Th2 cytokine production in ovalbumin-primed mice was inhibited by the administration of a neutralizing antibody to OX40-L from the time of first immunization with ovalbumin but no significant reduction in serum IgE or IgG1 was found.18 Our findings of a slight reduction in lymphoproliferation as measured by Cefodizime sodium spleen weight but no change in serum IgE concentrations with early anti-OX40-L treatment is in agreement with these observations. of COL12A1 Cefodizime sodium survivin, a protein involved in cell cycle progression and the inhibition of apoptosis.21 In common with CD28, OX40 activates nuclear factor (NF)-B22,23 with up-regulation of the antiapoptotic genes Bcl-xL and Bcl-2.24 Signalling through the PI3 kinase and P38MAP kinase pathways following OX40 ligation has been demonstrated to prolong the half lives of several cytokine mRNAs.25 There is evidence to suggest that Cefodizime sodium in addition to acting as a ligand for OX40, signals may be delivered to the B-lymphocyte by OX40-L mediating germinal centre formation26 and the differentiation of B lymphocytes into antibody-secreting cells.27 The observation that blockade of CD28 signalling becomes less effective at inhibiting HgCl2-induced autoimmunity when commenced after the initiation of the Th2 response and the concept that OX40 signalling follows sequentially from CD28 in Cefodizime sodium maintaining the activation of T lymphocytes led to the hypothesis that blockade of OX40 signalling would be an effective strategy for suppressing HgCl2-induced autoimmunity late in its course. Here we demonstrate that treatment with a monoclonal antibody to OX40-L early in the course of HgCl2-induced autoimmunity was ineffective but later treatment was suppressive. Materials and methods Animals Male BN rats weighing 250C350 g were purchased from Harlan Olac (Bicester, UK). Male rats were used because of their greater susceptibility to HgCl2-induced autoimmunity.28 All procedures were performed under halothane anaesthesia and were approved by the UK Home Office. Treatment with mercuric chloride HgCl2 (Sigma, Poole, UK) was dissolved at a concentration of 1 1 mg/ml in saline and was injected subcutaneously at a dose of 1 1 mg/kg for a total of five doses given on alternate days29. Humane end-points required killing of any animal with weight loss of more than 25%, severe ocular or oral mucositis, or arthritis affecting gait. Monoclonal antibodies ATM-2, a murine IgG1 antibody to rat OX40-L was prepared as described previously.8 Anti-CD80 (3H5) and anti-CD86 (24F) antibodies30 were prepared from tissue culture supernatant by ammonium sulphate precipitation and passage through a protein-A column. Both antibodies are murine IgG1. An isotype-matched control MOPC 21 (Sigma, St. Louis, MO) was prepared from clarified ascites by passage through a protein-A column. BN rats were injected intravenously with 100 g anti-OX40-L (033 mg/kg), 100 g each of anti-CD80 and anti-CD86 (033 mg/kg), or 100 g of MOPC 21 as an isotype control, in 1 ml 09% NaCl, initially daily for 3 days and then on alternate days until day 12 after the first HgCl2 injection (early treatment). Late treatment was by the same regimen, but commencing on day 8 after the first HgCl2 injection with the last dose on day 20. These doses were derived from preliminary dose-finding experiments. IgE enzyme-linked immunosorbent assay (ELISA) Serum was prepared from blood collected from a cut in the tail vein and stored at ?20 until assayed. Total IgE was measured by ELISA as described.28 Briefly, 96 well plates (Dynex Technologies Ltd, Billingshurst, UK) were coated with monoclonal anti-rat IgE heavy chain (Serotec Ltd, Oxford, UK) in carbonate buffer. Unoccupied binding sites were blocked with 5% skimmed milk in phosphate-buffered saline (PBS). Known concentrations of rat IgE myeloma protein (Serotec) or serum samples were added in duplicate to coated wells and singly to anti-IgE-free wells. Binding was detected with alkaline phosphatase-conjugated monoclonal anti-rat and light chain antibodies (Sigma) followed by = 12) for MOPC-treated animals and 101 g (096C108, = 12) for anti-OX40-L treated animals, MannCWhitney 0015. Normal BN rat spleens for animals weighing 250C350 g weighed 058 01 g (mean SD).3 There was no difference in the severity of caecal vasculitis on day 14 (data not shown). In a preliminary experiment an increase in the dose of anti-OX40-L to 500 g using the same protocol gave similar results. Open in a separate window Physique 1 Early treatment with anti-OX40-L antibody had no effect on serum IgE concentrations. Serum IgE concentrations are shown for.