R. D’Elia and K. J. Else treatment for collagen-induced arthritis (17). It is believed that E/S from helminths and ES-62 in particular can modulate the immune response in several ways including via interactions with B cells, macrophages and dendritic cells (18,19). Data suggest that exposure of macrophages to ES- 62 makes them unable to produce Th1/proinﬂammatory cytokines (20). In addition ES-62 drives the maturation of DCs and thus promotes the differentiation of naïve CD4+ T cells towards a Th2 phenotype (21). The ability of E/S to alter innate cell function is not uncommon and has been demonstrated for a number of other parasites (22,23). Interestingly both differentiated and undifferentiated intestinal pig epithelial cells predominantly produce IL-10 and IL-6 following stimulation with T. suis E/S (24). The ability of E/S to alter the biology of macrophages and dendritic cells (DC) is of particular interest given their involvement in the induction and maintenance of adaptive immune responses. Thus the response of DCs and/or macrophages to the S isolate E/S antigens may underlie the subsequent inability of the host to mount a protective Th2 response. In this study we explore the ability of E, J and S isolate E/ S to alter the phenotype and function of bone marrow-derived macrophages (BMMΦ) and bone marrow-derived dendritc cells (BMDCs). Cell surface expression of the co-stimulatory markers CD80, CD86 and CD40 along with MHC class II were analysed by ﬂow cytometry on BMMΦ and BMDCs after exposure to isolate-speciﬁc E/S. In addition the cytokine levels secreted by these APCs were analysed by cytokine bead array. We hypothesize that BMMΦ and or BMDCs would respond differently to S isolate E/S antigens compared to E/S from the E and J isolates at the level of costimulatory markers and/or cytokine production. MATERIALS AND METHODS Mice C57BL/6 male mice were obtained from Harlan (Bicester, UK) at 6–8 weeks of age. Mice were speciﬁc pathogen-free and maintained in sterile conditions in individually ventilated cages by the Biological Services Facility (BSF), University of Manchester, UK. All work was performed under the regulations of the Home Ofﬁce Scientiﬁc Procedures Act (1986). Parasites 124 The E, J and S isolates of T. muris were maintained as previously described (25). Excretory/secretory (E/S) antigen Parasite Immunology was collected by incubating adult worms in RPMI medium for 4 h. To conﬁrm that the E/S antigen was not contaminated with bacterial LPS or any other endotoxins a Limulus Amebocyte Lysate (LAL) Test Kit (Charles River Endosafe) was used (Levels for endotoxin in all samples of isolate E/S were all below 3 pg/mL). Previous data show that > 300 pg/mL of endotoxin is required to cause a cytokine response (26). Bone marrow macrophage culture BMMΦ were cultured as previously described (26). Brieﬂy bone marrow was recovered and pooled from the femurs of 3–5 naive C57BL/6 mice by ﬂushing the bone with Dulbecco’s modiﬁed Eagle’s medium (DMEM; Invitrogen, Paisley, UK) containing 10% fetal calf serum, 2 mm l -glutamine, 100 units/mL penicillin and 100 ng/mL streptomycin (complete DMEM). The cell suspension was passed through a 21 gauge needle, washed and cultured at 1 × 106/mL in complete DMEM containing 30% L929-conditioned media for 7 days. Macro- phage purity was assessed by staining with anti-CD11b-PE and anti-F4/80-FITC (BD Biosciences, Oxford, UK). Greater than 80% of live cells [measured by 7-amino- actinomycin D (Sigma) exclusion] were positive for either or both markers. Less than 1% of cells were positive for CD11c. Bone marrow dendritic cell culture Bone marrow was recovered and pooled from the femurs of 3–5 naïve C57BL/6 mice by ﬂushing the bone with complete medium (RPMI 1640 (Invitrogen, Carlsbad, CA) containing 5% foetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1% l -glutamine, 0·1% MTG (Sigma-Aldrich, St Louis, MO) on ice. The cell suspension was passed through a 21 gauge needle and washed. Cell con- centration was determined using a CASY-1 Cell Counter. DC culture followed a previous publication (27). Brieﬂy, following centrifugation the cell pellet was counted and resuspended at 2 × 106 cells/mL in 10 mL of RPMI supple- mented with 10% FCS, 100 U/mL Penicillin, 100 ng/mL Streptomycin, 2 mm l -glutamine and 0·5 mL of GM-CSF at 40 ng/mL (GM-CSF-containing supernatants were derived from Ag8 hybridoma cells (28)) and plated in bacteriologi- cal Petri dishes. A further 10 mL GM-CSF-containing medium was added after 3 days culture, and replaced after 6 days. After 7 days culture the non-adherent DCs were removed and purity was assessed by staining with anti-CD11b- PE and anti-CD11c-FITC (BD Biosciences, Oxford, UK). Greater than 65% of live cells [measured by (7AAD) 7- amino-actinomycin D (Sigma) exclusion] were positive for either or both markers. Fewer than 1% of cells were positive for F4/80. © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Parasite Immunology, 31, 123–131