Rapid Decrease of CD16 (FcγRIII) Expression on Heat-Shocked Neutrophils and Their Recognition by Macrophages
Figure 1
Delayed apoptosis in heat-shocked PMNs neutrophils. (a) Annexin V and propidium iodide staining of control, heat shocked (39°C or 41°C, 90 min) or apoptotic neutrophils. The percentage of cells in each subpopulation is shown in dot plots. Results of representative experiment of three performed are presented. (b) Expression of PS, annexin I and II on control, heat-shocked, or apoptotic neutrophils. (c) Effect of HS on the phagocytosis of neutrophils by hMDM. Phagocytosis of fresh, healthy neutrophils, preincubated at indicated temperatures (37–43°C, 90 min) measured immediately following preincubation (open bars) or after additional 24 hrs culture in standard conditions (gray bars). Neutrophils were added to macrophages at 2.5×106 cell/well of 24-well plate in medium with 10% human serum and incubated for 2 hrs at 37°C. Noningested neutrophils were removed by intensive washing of the hMDM monolayer. The cells were solubilized with detergent and the neutrophil elastase activity was measured in lysates as described in materials and methods. The intensity of phagocytosis is expressed as mOD/min of the substrate turnover catalyzed by neutrophil-derived elastase. (d) Heat shock inhibits DNA fragmentation in neutrophils. Freshly isolated neutrophils (control) or the cells treated for 90 min with heat shock were allowed to underwent spontaneous apoptosis for 24 hrs before DNA was isolated and subjected to electrophoresis. Results of a representative experiment out of three are presented in duplicates. (e) Preservation of the cell membrane integrity in heat-shocked neutrophils. The release of LTF, HNE, and LDH to the medium was measured following neutrophils treatment with heat shock for 90 min. The LTF, HNE, and LDH concentration in the culture medium of necrotic (sonicated) neutrophils was set as the positive control (100%). Data presented are mean (±SD). *,**, and ***, relative to controls, C.
