T cell hybridomas, B3Z86/90.14 (B3Z) and DOBW, were kindly provided by Dr. Nilabh Shastri (University of California, Berkeley, CA) and by Dr. Clifford V. Harding (Case Western Reserve University, Cleveland, OH), respectively (16,17). The DC cell line (DC2.4) was obtained from the Dana-Farber Cancer Institute, Boston, MA, USA (18).

DCs were generated from total BM cells as described previously (19). Briefly, BM cells obtained from femurs of BALB/c mmice were cultured in a 6-well plates (5×10⁶/well) in a culture medium supplemented with 200 U/ml rmGM-CSF. At days 3 and 4 from the initiation of the culture, nonadherent cells were discarded by replacing the culture medium with fresh medium containing the cytokines after gentle shaking. DCs were harvested by gentle pipetting at day 6.

Microspheres containing OVA were prepared using a solvent-evaporation method, as described previously (20), using OVA dissolved in 3% polyvinyl alcohol (4 mg/ml) and poly (DL-lactide-co-glycolide) (PLGA; lactide:glycolide=50:50; Sigma-Aldrich, St. Louis, MO, USA) dissolved in a mixture of acetone and ethanol (9:1) (5%). The concentration of OVA was determined by micro-bicinchoninic acid assay kit (Pierce, Rockford, IL) according to the manufacturer's instructions after lysing the microspheres in a lysis buffer containing 0.1% SDS and 0.1 N NaOH. For phagocytosis assays, micropheres containing both OVA and fluorescein isothiocyanate (FITC) were prepared by adding FITC (final, 5 mg/ml) to a mixture of acetone and ethanol (9:1) together with PLGA (final, 5%).

LacZ T cell activation assays were used to assess the amounts of cross-presented OVA peptides, as previously described (20). Briefly, DCs were cultured in the presence of different concentrations of aspirin (Sigma-Aldich) or ibuprofen (Sigma-Aldich) for 18 h in 96-well plates (1×10⁵/well), and then added with OVA-microspheres (50µg/ml as OVA). After 2 h incubation at 37℃, the plate was washed twice with 300µl/well of pre-warmed PBS, and then fixed with 100µl/well of ice-cold 1.0% paraformaldehyde for 5 min at room temperature. The plate was washed 3 times with 300µl/well of PBS, and B3Z cells were added (2×10⁵/well). After incubating for 4 h at 37℃, lacZ activity was measured either by colorimetric analysis after incubating freeze-thaw lysed cells with β-galactosidase substrate, chlorophenol red β-D-galactopyranoside (Calbiochem, Darmstadt, Germany), as described previously (20).

BM-DCs were cultured in the presence of different concentrations of aspirin or ibuprofen for 18 h in 96-well plates (1×10⁵/well), and then added with OVA-microspheres (50µg/ml as OVA). After 2 h incubation at 37℃, unphagocytized OVA-microspheres were removed by suction, and then fixed with ice-cold 1.0% paraformaldehyde for 5 min at room temperature. The plate was then washed twice with 300 µl/well of pre-warmed media, and added with DOBW cells (1×10⁵/well). After 24 h incubation at 37℃, the plate was centrifuged at 1,800 rpm, and the culture supernatant was collected and assayed for IL-2 content using an IL-2 ELISA kit (BD Biosciences, San Jose, CA).

DCs were cultured in the presence of different concentrations of aspirin or ibuprofen for 18 h in 6-well plates (2×10⁶ cells/well), and then added with microspheres (average diameter, 300 nm) containing both ovalbumin (OVA) and fluorescein isothiocyanate (FITC). After 2 h, unphagocytozed microspheres were removed by washing with pre-warmed PBS. The plate was chilled on ice for 20 min, then the cells were harvested by treating with Cell stripper solution (Cellgro Mediatech, Herndon, VA) as suggested in the manufacturer's instruction, fixed in 1% paraformaldehyde in PBS, and flow cytometric analysis was performed on a FACS Calibur flow cytometer (Becton Dickinson).

DCs were cultured in the presence of different concentrations of aspirin or ibuprofen for 18 h in 6-well plates (2×10⁶ cells/well). The plate was then chilled on ice for 20 min, and the cells were harvested by treating with Cell stripper solution (Cellgro Mediatech). The cells were stained with monoclonal antibodies recognizing murine cell surface molecules after blocking of FcR-binding anti-CD16/CD32 monoclonal antibody (clone 2.4G2), and flow cytometric analysis was performed on a FACS Caliver (Becton-Dickinson). The monoclonal antibodies, anti-H-2K^b (clone AF6-88.5), anti-I-A^b (clone AF6-120.1), anti-CD40 (clone 3/23), anti-CD54 (clone 3E2), anti-B7-1 (clone 16-10A1), anti-B7-2 (clone GL1), and isotype-matched control antibodies were purchased from BD Biosciences.

To examine the effects of the aspirin and ibuprofen on the cross-presentation, DC2.4 cells were cultured in the presence of the drugs for 18 h, and then allowed to phagocytose OVA-microspheres for 2 h. The DC2.4 cells were then washed, fixed with paraformaldehyde, and the amount of OVA peptide-class I MHC complexes was measured using a T cell hybridoma, B3Z, which recognizes OVA peptide (SIINFEKL)-H-2K^b complex and expresses β-galactosidase (16). As shown in Fig. 1, aspirin inhibited MHC class I-restricted OVA presentation at concentrations of 500µM or above. The inhibitory activity of ibuprofen on MHC class I-restricted OVA presentation was observed at concentrations of 250µM or above.

The effects of the drugs on the MHC class II-restricted presentation of exogenous OVA were examined in DCs generated from bone marrow cells (BM-DCs) with GM-CSF. BM-DCs were treated with different concentrations of the drugs for 18 h, and then allowed to phagocytize OVA-microspheres for 2 h. The DCs were then washed, fixed with paraformaldehyde, and then the amount of OVA peptide-class II MHC complexes was measured using OVA-specific CD4 T cell hybridoma, DOBW cells. As shown in Fig. 2, aspirin inhibited MHC class II-restricted OVA presentation at concentration of 250µM or above. The inhibitory activity of ibuprofen on MHC class II-restricted OVA presentation was observed at concentrations of 125µM or above.

To examine the effects of aspirin and ibuprofen on the acquisition of antigen presenting function, the drugs were added to DC differentiation-inducing cultures at different time points, days 0, 3 and 5 after the initiation of the culture. BM-DCs were harvested on day 6 from the initiation of the culture, and the ability to present exogenous antigen in association with MHC class II molecules was examined as in Fig. 2. As shown in Fig. 3A, DCs generated from mouse bone marrow cells in the presence of aspirin from the initiation of the culture (Day0) or 3 days after the initiation of the culture (Day3) were profoundly suppressed in MHC class II-restricted OVA presenting capability. This inhibitory effect of aspirin appeared to be dose dependent. When developing DCs were exposed to aspirin at a later time point (Day5, 5 days after the initiation of culture), the suppressive activity of aspirin on antigen presenting capability was weaker compared to the earlier exposures (Day0 and Day3). Likewise, DCs generated in the presence of ibuprofen from earlier time points of differentiation (Day0 and Day5) were significantly suppressed in MHC class II-restricted antigen presenting capability. We also found that DCs generated in the presence of aspirin or ibuprofen were suppressed in the MHC class I-restricted exogenous antigen presentation, and the suppressive activities of the drugs were more prominent when developing DCs were exposed to the drugs at earlier time points (data not shown).

To examine whether the suppressed capacity of aspirin or ibuprofen-treated DCs to present OVA peptides in association with MHC molecules was due to decreased phagocytic activity, DC2.4 cells were cultured with the drugs (1 mg/ml) for 18 h, and then added with microspheres containing both OVA and FITC. After 2 h incubation, unphagocytized microspheres were washed, and the cells were harvested by gentle pipetting after cooling on ice. Flow cytometric analysis of the harvested cells showed that neither of the drugs suppressed the phagocytic activity of DC2.4 cells (Fig. 4).

To examine whether the suppressed capacity of aspirin or ibuprofen-treated DCs to present OVA peptides in association with MHC molecules was due to the inhibition of expression of MHC molecules on the cell surface, DC2.4 cells were cultured with the drugs (1 mg/ml) 18 h, and then the expression levels of MHC class I and class II molecules were determined by anti-H-2K^b and anti-I-A^b monoclonal antibodies. As shown in Fig. 5, aspirin did not affect the expression of MHC class I or class II molecules. Ibuprofen, which inhibited MHC-restricted exogenous OVA presentation, even slightly increased the expression of MHC class I and class II molecules. Flow cytometric analysis for major co-stimulatory molecules such as B7-1, B7-2, and CD40 also showed that aspirin did not affect the expression of these co-stimulatory molecules (Fig. 6). Again, ibuprofen, which inhibited MHC-restricted exogenous OVA presentation, even slightly increased the expression of co-stimulatory molecules such as B7-1, B7-2, and CD40 (Fig. 6).