Dexamethasone (Dex) was shown to inhibit the differentiation maturation and antigen-presenting function of dendritic cells (DC) when added during DC generation or maturation stages. that Dex inhibits intracellular processing events of phagocytosed antigens in macrophages. by enriching tolerogenic macrophages while inducing apoptosis of effector T cells (12 13 14 Dex was also shown to severely impair the differentiation maturation and function of dendritic cells (DCs) and macrophages (15 16 17 The effects of Dex on DCs and macrophages however were investigated in cells cultured in the presence of Dex for two to several days. In the present study we examined Ondansetron HCl the direct effects of Dex around the MHC-restricted presentation of exogenous antigens. Macrophages were generated from mouse bone marrow cells and allowed to phagocytose microencapsulated ovalbumin (OVA) in the presence of Dex for 2 h. The efficacy of OVA peptide presentation was evaluated using OVA-specific CD8 and CD4 Ondansetron HCl T cells. Our results show that Dex inhibits the intracellular processing events of phagocytosed antigens in macrophages. We also discovered that immature macrophages are much more sensitive to the Dex-induced inhibition of MHC-restricted antigen processing than mature macrophages. MATERIALS AND METHODS Cell lines Ondansetron HCl and reagents The T-cell hybridoma cell lines B3Z86/90.14 (B3Z) and DOBW were kindly provided by Dr. Nilabh Shastri (University or college of California Berkeley CA USA) and Dr. Clifford V. Harding (Case Western Reserve University or college Cleveland OH USA) respectively (18 19 Recombinant human M-CSF was purchased from PeproTech (Rocky Hill NJ USA). Dexamethasone was purchased from Sigma-Aldrich (St. Ondansetron HCl Louis MO USA). Generation of macrophages from bone marrow cells Macrophages were generated from mouse bone marrow using recombinant human macrophage colony stimulating factor (rhM-CSF). Briefly bone marrow cells obtained from femurs of C57BL/6 or Balb/c mice were cultured in a 6-well plate (5×106/well) in culture media supplemented with 20 U/ml rhM-CSF. At days 3 and 4 after the initiation of Rabbit Polyclonal to NRIP3. the culture non-adherent cells were discarded by gentle shaking and replacement of the culture medium with new medium made up of rhM-CSF. Immature macrophages were harvested on day 6 using cell stripper answer. Lipopolysaccharide (100 ng/ml) was added to immature macrophage cultures for maturation. Cells were cultured for 2 additional days and Ondansetron HCl then harvested using cell stripper answer. Preparation of OVA-nanospheres Nanospheres made up of OVA were prepared using a homogenization/solvent evaporation method with 400μl of OVA-containing water (50 mg/ml OVA) and 2 ml of ethyl acetate made up of poly(lactic-co-glycolic acid) (100 mg/ml Sigma-Aldrich) as explained previously (Lee et al. 2010 Fluorescein isothiocyanate (FITC)-made up of PLGA-nanospheres were prepared by adding FITC to the ethyl acetate phase together with PLGA. The OVA content was determined using a micro-bicinchoninic acid assay kit (Pierce Rockford IL USA) after lysis of the nanospheres with a lysis buffer made up of 0.1% SDS and 0.1 N NaOH. MHC class I-restricted presentation assay Class I MHC-complexed OVA peptide quantities on macrophages were assessed using B3Z cells (20). Briefly macrophages (1×105/well) generated from bone marrow cells of C57BL/6 mice (H-2b) were incubated with the indicated amounts of Dex for 2 h and then OVA-nanospheres were added (50μg as OVA). After 2 h incubation at 37℃ the plate was washed twice with pre-warmed PBS (300μl/well) and then fixed with ice-cold 1.0% paraformaldehyde (100μl/well) for 5 min at room temperature followed by washing of the plate three times with PBS (300μl/well). Class I MHC-complexed OVA peptide quantities were assessed by IL-2 secretion assays after culturing the paraformaldehyde-fixed macrophages with CD8.OVA cells (2×104/well) Ondansetron HCl for 18 h as described previously (20). MHC class II-restricted presentation assay Class II MHC-complexed OVA peptide quantities on macrophages were assessed using DOBW cells (20). Briefly macrophages (1×105/well) generated from bone marrow cells of BALB/C mice (H-2d) were incubated with the indicated amounts of Dex for 2 h and then OVA-nanospheres were added (50μg as OVA). After 2 h incubation at 37℃ unphagocytosed nanospheres were removed by suction and then fixed with ice-cold 1.0% paraformaldehyde (100μl/well) for 5 min at.