Figure. 4. Engagement of CD11b N-linked glycans regulates PMN phagocytosis, ROS release and apoptosis.

Human PMN were incubated with 10μg/ml MMR (A), 10μg/ml GNA (B) or 10μg/ml PHA-E (C) for 60 minutes at 37^oC before fluorescent microsphere phagocytosis/uptake was quantified by measuring changes in fluorescence by flow cytometry. (D) Data is mean fluorescence intensity normalized to non-treated PMN and is expressed as mean ± SEM. n = 3 independent donors *, p<0.05, **, p<0.01. (E) Human PMN were incubated with 10μg/ml PHA-E, 10μg/ml GNA or 10μg/ml MMR before treatment with 100μM cytochrome C. Reduction of cytochrome C in response to 500nM fMLF was measured by quantifying changes in absorbance at 550nm at 2,5,10,15 and 20 minutes. Data is fold change in absorbance relative to time zero and is expressed as ± SEM. n = 3 independent donors **, p<0.01, ***, p<0.001. (F) For apoptosis assays, human PMN were incubated with 10μg/ml PHA-E, 10μg/ml GNA or 10μg/ml MMR before assessment of surface expression of Annexin V and 7AAD by flow cytometry. Cells negative for Annexin IV and 7AAD were considered non-apoptotic. Representative flow plots show percentage apoptotic PMN under the defined conditions. (G) Quantification of data from flow plots. All Annexin-V positive cells were considered early apoptotic neutrophils. Data is expressed as mean ± SEM. n = 4 independent donors *, p<0.05, **, p<0.01.