American Society of Hematology

Figure 1.

$Figure 1. A fraction of monocyte-derived DCs exhibits a mature phenotype after exposure to PR3 but not HLE. Immature DCs cultured for 6 days in IL-4 and GM-CSF were stimulated with PR3, HLE, CG, and TNF-α. After 48 hours, the DCs were stained for CD14, CD80, CD83, CD86, and HLA-DR and analyzed by flow cytometry. Isotype-matched antibodies served as control in all experiments. (A) Dose-response curves for PR3, HLE, and CG; data are presented as mean percentage of positive cells. (B) Expression of CD14, CD80, CD83, CD86, and HLA-DR. The concentration of proteases used (10 μg/mL) is equivalent to the top of the dose-response curve. Data are presented as mean MFI ratios ± SEM. *P < .05; **P < .03; ***P < .001 (Bonferroni test). The experiments pertaining to panels A-B were performed separately. (C) To understand whether the enzymatically active PR3 was required for these effects on DCs, the cells were incubated for 24 hours with enzymatically active PR3, enzymatically inactive mutated PR3, and PMSF and PMSF-pretreated PR3. The expression of CD83 on the cell surface was analyzed by flow cytometry. The histograms show fluorescence values on gated cells. The bold lines represent isotype Abs control. Results are representative of 1 of 3 independent experiments performed in duplicate.$

A fraction of monocyte-derived DCs exhibits a mature phenotype after exposure to PR3 but not HLE. Immature DCs cultured for 6 days in IL-4 and GM-CSF were stimulated with PR3, HLE, CG, and TNF-α. After 48 hours, the DCs were stained for CD14, CD80, CD83, CD86, and HLA-DR and analyzed by flow cytometry. Isotype-matched antibodies served as control in all experiments. (A) Dose-response curves for PR3, HLE, and CG; data are presented as mean percentage of positive cells. (B) Expression of CD14, CD80, CD83, CD86, and HLA-DR. The concentration of proteases used (10 μg/mL) is equivalent to the top of the dose-response curve. Data are presented as mean MFI ratios ± SEM. *P < .05; **P < .03; ***P < .001 (Bonferroni test). The experiments pertaining to panels A-B were performed separately. (C) To understand whether the enzymatically active PR3 was required for these effects on DCs, the cells were incubated for 24 hours with enzymatically active PR3, enzymatically inactive mutated PR3, and PMSF and PMSF-pretreated PR3. The expression of CD83 on the cell surface was analyzed by flow cytometry. The histograms show fluorescence values on gated cells. The bold lines represent isotype Abs control. Results are representative of 1 of 3 independent experiments performed in duplicate.

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