Intraphagosomal measurement of the magnitude and duration of the oxidative burst

Abstract

Generation of an oxidative burst within the phagosomes of neutrophils, dendritic cells and macrophages is an essential component of the innate immune system. To examine the kinetics of the oxidative burst in the macrophage phagosome, we developed two new assays using beads coated with oxidation-sensitive fluorochromes.These assays permitted quantification and temporal resolution of the oxidative burst within the phagosome. The macrophage phagosomal oxidative burst is short lived,with oxidation of bead-associated substrates reaching maximal activity within 30 min following phagocytosis.Additionally, the extent and rate of macrophage phagosomal substrate oxidation were subject to immunomodulation by activation with lipopolysaccharide and/or interferon-gamma.

Figure 1. H2HFF-OxyBURST fluorescence is pH insensitive

Fluorescence ratio of pre-oxidized H2HFF-OxyBURST beads were acquired in buffers of various pH and plotted as calibrated fluorescence using the equation CF = OB/AF, where CF is calibrated fluorescence, OB is H2HFF-OxyBURST fluorescence, and AF is Alexa594 fluorescence. The trace represents the averaged endpoint values over three repetitions, error bars represent SD.

Figure 2. Phagosomal H2HFF-OxyBURST oxidation profiles

H2HFF-OxyBURST oxidation kinetics were plotted as calibrated fluorescence using the equation CF = OB/AF. Measurements were taken every 2 seconds for 60 minutes. Activation was accomplished by LPS (10ng/ml) addition 15 hr prior to experimentation; the trace represents the averaged values over three repetitions, error bars represent SD. B. Beads were coated in 1.0 mg or 0.5 mg H2HFF-OxyBURST and evaluated in LPS activated macrophage phagosomes. Maximal oxidation is plotted as calibrated fluorescence using the equation CF = OB/AF. Activation was accomplished by pretreatment with LPS (10 ng/ml) 15 hr prior to phagocytosis. The trace represents the averaged values over two repetitions, error bars represent SD.

Figure 2. Phagosomal H2HFF-OxyBURST oxidation profiles

H2HFF-OxyBURST oxidation kinetics were plotted as calibrated fluorescence using the equation CF = OB/AF. Measurements were taken every 2 seconds for 60 minutes. Activation was accomplished by LPS (10ng/ml) addition 15 hr prior to experimentation; the trace represents the averaged values over three repetitions, error bars represent SD. B. Beads were coated in 1.0 mg or 0.5 mg H2HFF-OxyBURST and evaluated in LPS activated macrophage phagosomes. Maximal oxidation is plotted as calibrated fluorescence using the equation CF = OB/AF. Activation was accomplished by pretreatment with LPS (10 ng/ml) 15 hr prior to phagocytosis. The trace represents the averaged values over two repetitions, error bars represent SD.

Figure 3. Activation enhances phagosomal H2HFF-OxyBURST oxidation

A. H2HFF-OxyBURST oxidation kinetics were plotted as calibrated fluorescence using the equation CF = OB/AF. Activation was accomplished by pretreatment with LPS (10ng/ml) or INF-γ (100 U/ml) 15 hr prior to phagocytosis. For synergistic activation cells were treated with INF-γ (100 U/ml) for 13 hr and then LPS (10ng/ml) 2 hr prior to phagocytosis. The trace represents the averaged values over three repetitions, error bars represent SD.

Figure 4. Phagosomal Bodipy^581/591 bead oxidation profiles

A. Bodipy^581/591 bead oxidation kinetics was plotted with the equation CF = GF/RF, where CF is calibrated fluorescence, GF is the green Bodipy^581/591 signal, and RF is the red Bodipy^581/591 signal. Measurements were taken every 20 seconds for 60 minutes. Activation was accomplished by treatment with LPS (10ng/ml) 15 hr prior to phagocytosis. The kinetic trace is from a representative experiment and is the average of 10 phagosomal bead values from each condition, error bars represent SD. B. Serial confocal images of phagosomal Bodipy^581/591 beads in resting macrophages. Scale bar; 5 μm.

Figure 4. Phagosomal Bodipy^581/591 bead oxidation profiles

A. Bodipy^581/591 bead oxidation kinetics was plotted with the equation CF = GF/RF, where CF is calibrated fluorescence, GF is the green Bodipy^581/591 signal, and RF is the red Bodipy^581/591 signal. Measurements were taken every 20 seconds for 60 minutes. Activation was accomplished by treatment with LPS (10ng/ml) 15 hr prior to phagocytosis. The kinetic trace is from a representative experiment and is the average of 10 phagosomal bead values from each condition, error bars represent SD. B. Serial confocal images of phagosomal Bodipy^581/591 beads in resting macrophages. Scale bar; 5 μm.