ADAM10 and ADAM17 promote downregulation of CD16 by proteolytic cleavage. (A) Broad-spectrum metalloproteinase inhibitors prevent downregulation of CD16 on slanDCs. Freshly isolated slanDCs were cultured for 6 hours in the presence of broad-spectrum metalloproteinase inhibitors with distinct specificities for certain MMPs and ADAMs (supplemental Table 1). Cultured controls did not contain an inhibitor. Uncultured controls were stored on ice. After cell culture, CD16 expression was measured by flow cytometry. Expression of CD16 is depicted as percent expression of uncultured slanDCs. (B) Inhibiting ADAM10 and ADAM17 activity with specific inhibitors prevents downregulation of CD16 on slanDCs. slanDCs were cultured for 8 hours in the presence of GW280264x, blocking both ADAM10 and ADAM17, and GI254023x, preferentially blocking ADAM10. Controls did not contain an inhibitor or did contain the inhibitor’s solvent (DMSO). Uncultured control cells were left on ice. CD16 expression was measured by flow cytometry and is depicted as percent expression of uncultured slanDCs. The histograms above the diagram show the CD16 staining of 1 representative experiment. (C) Soluble CD16 is produced in cell cultures of freshly isolated slanDCs but is hardly produced in the presence of metalloproteinase inhibitors. 1 × 106 slanDCs, monocytes, and CD4+ T cells, as well as slanDCs in the presence of 50 µM Ro327315 and 50 µM GW280264x, were incubated for 1 hour at 37°C. Supernatants of the cell cultures were then analyzed by Western blot for the presence of soluble CD16. (D) Preventing loss of CD16 prevents loss of IC binding. Freshly isolated slanDCs were cultured without the addition of metalloproteinase inhibitors, in the presence of GW280264x and the inhibitor’s solvent (DMSO). Control cells were stored on ice. After 8 hours of cell culture, binding of fluorescent ICs was quantified by flow cytometry and is depicted as percent IC binding of uncultured slanDCs. The histograms above the diagram are results of 1 representative experiment. Upper row: Light gray histograms show the control staining; dark gray histograms represent IC binding. ΔMFI is calculated by subtraction of the MFI of the control staining from the MFI of the samples with bound ICs. Lower row: CD16 expression of the respective slanDCs. Data represent mean ± SEM (N = 5).
