Dissociation Between Respiratory Burst Activity and Deoxyglucose Uptake in Human Neutrophil Granulocytes: Implications for Interpretation of 18F-FDG PET Images

Dissociation Between Respiratory Burst Activity and Deoxyglucose Uptake in Human Neutrophil Granulocytes: Implications for Interpretation of ¹⁸F-FDG PET Images

Hazel A. Jones, PhD¹, Karen A. Cadwallader, PhD¹, Jessica F. White, MA¹, Mohib Uddin, BSc¹, A. Michael Peters, MD² and Edwin R. Chilvers, PhD¹

¹ Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, United Kingdom
² Department of Radiology, University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, United Kingdom

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FIGURE 1. TNF ${alpha}$ priming of fMLP-stimulated O₂^- generation and ³H-DG uptake in human neutrophils. (A) Measurement of O₂^- release by superoxide dismutase-inhibitable reduction of cytochrome C during 10-min incubation with fMLP (100 nmol/L) after priming with TNF ${alpha}$ (200 U/mL, 30 min). Data points represent mean ± SEM of 3 separate experiments, each performed in triplicate. (B) ³H-DG uptake in TNF ${alpha}$ (200 U/mL, 30 min)-primed neutrophils. After priming, or incubation with PBS, cells were stimulated with fMLP (100 nmol/L) or PMA for indicated time. Data shown are for control unprimed cells ( ${circ}$ ), TNF ${alpha}$ -primed (200 U/mL, 30 min) unstimulated cells ( ${square}$ ), unprimed fMLP (100 nmol/L)-stimulated cells (•), TNF ${alpha}$ -primed fMLP-stimulated cells ( ${blacksquare}$ ), and PMA ( ${triangleup}$ ). Results are mean ± SEM of 5 separate experiments. (C) Quantification of O₂^- release with lucigenin-dependent chemiluminescence monitored at 9-s-cycle intervals in control unprimed cells ( ${circ}$ ), unprimed fMLP (100 nmol/L)-stimulated cells (•), and TNF ${alpha}$ -primed fMLP-stimulated cells ( ${blacksquare}$ ). Data are from single experiment, representative of 3 separate experiments.

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FIGURE 2. (A). Superoxide anion release from control unstimulated cells. Cells were primed with TNF ${alpha}$ (200 U/mL) for 30 min, stimulated with fMLP (100 nmol/L) for 10 min, and treated with 100 nmol/L PMA for 30 min. O₂^- release was measured by the superoxide dismutase-inhibitable reduction of cytochrome C. Data points represent mean ± SEM of 3 separate experiments, each performed in triplicate. (B) Effect of PMA concentration on ³H-DG uptake. Cells were incubated with ³H-DG; radioactivity in cells was measured at 30 min.

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FIGURE 3. Effect of PI3-kinase inhibitor wortmannin on O₂^- release, degranulation, and DG uptake. (A) Human neutrophils were incubated with vehicle (0.01% DMSO) (open bars) or 100 nmol/L wortmannin (filled bars) for 10 min before TNF ${alpha}$ priming (200 U/mL for 30 min) and fMLP stimulation (100 nmol/L, 10 min). O₂^- release was quantified by superoxide dismutase-inhibitable reduction of cytochrome C. Data points represent mean ± SEM of 3 separate experiments, each performed in triplicate. (B) Wortmannin inhibits neutrophil degranulation. fMLP-induced MPO release in TNF ${alpha}$ -primed human neutrophils was performed as described. Human neutrophils (12.5 x 10⁶/mL) were suspended in PBS with Ca²⁺ and Mg²⁺ and treated with TNF ${alpha}$ (200 U/mL) or PBS at 37°C for 30 min. Cells were then incubated with 100 nmol/L wortmannin (filled bars) or vehicle (0.01% DMSO) (open bars) for 10 min before stimulation with fMLP (100 nmol/L) or PBS. Reaction was terminated at 10 min by addition of ice-cold PBS and then immersing tubes in ice. MPO release was quantified by spectrophotometric assessment (460 nm) as described previously (15). Data represent mean ± SEM of 3 separate experiments, each performed in triplicate. (C) Wortmannin does not affect ³H-DG uptake. Human neutrophils (12.5 x 10⁶/mL) were suspended in PBS with Ca²⁺ and Mg²⁺ and treated with TNF ${alpha}$ (200 U/mL) or PBS at 37°C for 30 min. Cells were then incubated with 100 nmol/L wortmannin (filled bars) or vehicle (0.01% DMSO) (open bars) for 10 min before incubation with ³H-DG and stimulation with fMLP (100 nmol/L) or PBS. Data are shown for uptake over 30 min. dpm = disintegrations per minute.

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FIGURE 4. Wortmannin has no effect on fMLP-stimulated change in neutrophil shape. Neutrophils were incubated with increasing concentrations of fMLP with ( ${circ}$ ) or without (•) wortmannin (100 nmol/L). After addition of glutaraldehyde, shape of cells was visually assessed for change using light microscope. Data represent mean ± SEM of 3 separate experiments, each performed in triplicate.