Article Figures & Data
Figures
- FIGURE 1.
Monokine secretion induced by solid-phase mAbs to HLA. A, DT13.2 and BC20.7 were cultured in the presence of Der f 1 peptide (for DT13.2) or BCGa peptide (for BC20.7) and irradiated autologous PBMC, with or without anti-class II HLA mAbs. B, Adherent cells were incubated at 6 × 104 cells/well where 10 μg/ml of 1a3 (▪) and mouse IgG2a (□) are immobilized at 37°C in a CO2 incubator. Culture supernatants were collected at the indicated time points.
- FIGURE 2.
Activation of MAP kinases by mAbs to class II HLA. A, Monocytes were cocultured for 16 h on anti-DR (L243)-coated plates, with the indicated inhibitors, at the indicated concentrations. Culture supernatants were collected and stored in aliquots at −80°C until determinations of cytokine concentrations. One hundred percent IL-1β production was 205 pg/ml. Viable cell contents were determined using trypan blue. B, After 10 and 60 min of stimulation with solid-phase mAbs (anti-DR L243, anti-DQ 1a3, and anti-DP B7/21), monocytes were lysed and subjected to Western blot analysis either with Abs specific for Erk and p38 or with the activated form of Erk and p38. C, Relative densities are shown based on B.
- FIGURE 3.
Fc and TNF-α are not involved in Erk and IL-1β activation induced by class II ligation. A, Ab preparations including F(ab′)2 of L243 were used for stimulation of monocytes, as for Fig. 2B. B, Anti-TNF-α at 10 μg/ml and controls were added to this culture system. C, TNF-α at indicated concentrations were cocultured with monocytes for 16 h in the presence of anti-TNF-α or control mIgG1 at 10 μg/ml, and IL-1β levels in culture supernatant fluids were determined.
- FIGURE 4.
Effect of PD98059 and SB203580 on IL-10 and IL-1β secretion. A–C, Peptide-pulsed monocytes were precultured for 60 min with the indicated inhibitors, at the indicated concentrations, and cocultured with emetine-treated T cells (BC20.7) for 24 h. Culture supernatants were collected and stored in aliquots at −80°C until determinations of cytokine concentrations. One hundred percent IL-1β production was 115 pg/ml. D, Cell viability (for the highest concentrations of the inhibitors shown in B and C) was determined by trypan blue exclusion. Results were expressed as means ± SD of triplicate cultures. ∗, p < 0.05.
- FIGURE 5.
Summary of class II HLA-mediated MAP kinase (MAPK) activation. Note that other class II signaling elements that can be additive or modify the signaling via MAP kinases are not illustrated.
- FIGURE 6.
Restriction molecules and cytokine production patterns of short-term T cell lines. Der f (crude mite Ag)-specific short-term T cell lines (A), PPD-specific short-term T cell lines (B), and X19-reactive T cell clones (C) of various restriction patterns were restimulated with excess concentrations of Ags (A and B, 10 μg/ml; C, 500 μM) and then after a 48-h incubation, culture supernatants were collected for measurements of IFN-γ and IL-4 production by ELISA. One spot indicates one cell line.
Tables
- Table I.
Monokine production from monocytes stimulated with emetine-treated T cells + peptidea
Monokine T Cell Clones (HLA Restriction) for Stimulation (pg/ml) BC20.7 (DR14) DT13.2 (DQ6) OT1.1 (DP5) IL-1β 105.0 24.0 21.0 IL-6 87.5 62.5 140.0 IL-10 172.5 787.5 725.0 IL-18 <15.0 <15.0 <15.0 IL-12 (p40+ p70) 145.0 75.0 20.5 GM-CSF 475.0 1150.0 887.5 TNF-α 887.5 642.5 230.0 IL-10 / IL-1β 1.6 32.8 34.5 a Emetine-treated T cells (BC20.7, DT13.2, and OT1.1) were cultured with peptide-prepulsed monocytes. The concentration of the peptides for each clonal response was 625-fold as much as the ED50 (5, 112.5, and 62.5 μM for BC20.7, DT13.2, and OT1.1, respectively). Culture supernatants after a 16-h (for IL-12), 24-h (for IL-1β, IL-10, IL-18, GM-CSF, and TNF-α), and 48-h (for IL-6) incubation were collected and subjected to ELISA. Results are expressed as the mean value of triplicate determinations. SE was <20%.
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