Rapid Decrease of CD16 (FcγRIII) Expression on Heat-Shocked Neutrophils and Their Recognition by Macrophages
Figure 7
Geldanamycin and radicicol enhance the anti-inflammatory effect of HS neutrophils which is associated with decrease of CD16 expression. (a) Neutrophils were incubated initially for 30 min at 37°C with or without (C) inhibitors of HSP90: geldanamycin (Ge) or radicicol (Rad) and then for additional 90 min at 41°C (HS). For enzymatic removal of surface CD16 PMNs were incubated with PI-PLC (0.5 IU/mL) for 45 min at 37°C in culture medium without serum. The enzymatic reaction was stopped by serum addition. For inhibition of spontaneous exocytosis PMNs were incubated with Rac inhibitor NSC23766 (50 μM) for 15 min at 37°C in culture medium. Finally, cells were centrifuged, resuspended in fresh culture medium and added to macrophages. Following 1 hr of co-incubation cytokine production was stimulated with 10 ng/mL LPS (E. coli, 0127:B8) for 4 hrs. TNF-α was subsequently measured in cell-free culture supernatants (open bars). Prior to addition to macrophages an aliquot of neutrophils was subjected to flow cytometric (MFI) measurement of CD16 expression (gray bars). (b) The anti-inflammatory effect of neutrophils was induced by preincubation of PMNs with CD16-specific antibodies (clones Leu11b, 3G8, Dj130c and GRM1) for 15 min at 20°C in culture medium. Following the preincubation neutrophils were resuspended in fresh culture medium and added to macrophages. Results (TNF-α) are expressed as percent (mean ± SD) of positive control that is hMDMs stimulated with LPS. Results (CD16) are expressed as percent (mean ± SD) of positive control that is freshly isolated neutrophils. *,***, relative to positive control.
