Figure 4.
Norbin-deficient neutrophils have an increased ability to kill bacteria, in a ROS-dependent manner, and increased ROS production in response to pathogens. Isolated neutrophils of the indicated genotypes were primed with 50 ng/mL TNF-α, 20 ng/mL GM-CSF before incubation with serum-opsonized S pneumoniae at a ratio of 1:1 (A) or 1:2 (C) for 15 minutes. Samples were sonicated in 0.05% saponin and serial dilutions cultured on blood agar to quantify CFU. Isolated neutrophils primed as in panel A were incubated with serum-opsonized S aureus at a ratio of 1:1 (B) or 1:2 (D) bacteria to neutrophils for 15 minutes. Panel D includes the indicated concentrations of the ROS inhibitor diphenyleneiodonium (DPI). CFU were determined as in panel A except that samples were cultured on luria broth agar. Data are mean ± standard error of the mean of 3 independent experiments for panel A, 5 to 9 for panel B, 4 for panel C, and 7 for panel D; each dot is the mean of 1 experiment performed in duplicate. The right-hand part of panel D shows samples without neutrophils. Statistics comprised one-way analysis of variance with Tukey’s multiple comparison test (panels A and B), paired two-tailed Student t test (panel C), and two-way analysis of variance with Šidák’s multiple comparisons test (panel D). (E) ROS production by isolated neutrophils from mice of the indicated genotypes, measured by using real-time chemiluminescence assay with luminol and horseradish peroxidase for extracellular and intracellular ROS, stimulated with serum-opsonized heat-inactivated S pneumoniae (200 bacteria/neutrophil), serum-opsonized live S aureus (5 bacteria/neutrophil), or zymosan (3 particles/neutrophil), as indicated, and quantified as area under the curve (AUC). Mock-stimulated ROS production was subtracted and is not shown for clarity. Data are mean ± standard error of the mean of the indicated numbers of independent experiments; each dot is the mean AUC from 1 experiment. Statistics comprised one-way analysis of variance with Holm-Šidák’s (S pneumoniae, S aureus) or Tukey’s (zymosan) multiple comparisons tests.

Norbin-deficient neutrophils have an increased ability to kill bacteria, in a ROS-dependent manner, and increased ROS production in response to pathogens. Isolated neutrophils of the indicated genotypes were primed with 50 ng/mL TNF-α, 20 ng/mL GM-CSF before incubation with serum-opsonized S pneumoniae at a ratio of 1:1 (A) or 1:2 (C) for 15 minutes. Samples were sonicated in 0.05% saponin and serial dilutions cultured on blood agar to quantify CFU. Isolated neutrophils primed as in panel A were incubated with serum-opsonized S aureus at a ratio of 1:1 (B) or 1:2 (D) bacteria to neutrophils for 15 minutes. Panel D includes the indicated concentrations of the ROS inhibitor diphenyleneiodonium (DPI). CFU were determined as in panel A except that samples were cultured on luria broth agar. Data are mean ± standard error of the mean of 3 independent experiments for panel A, 5 to 9 for panel B, 4 for panel C, and 7 for panel D; each dot is the mean of 1 experiment performed in duplicate. The right-hand part of panel D shows samples without neutrophils. Statistics comprised one-way analysis of variance with Tukey’s multiple comparison test (panels A and B), paired two-tailed Student t test (panel C), and two-way analysis of variance with Šidák’s multiple comparisons test (panel D). (E) ROS production by isolated neutrophils from mice of the indicated genotypes, measured by using real-time chemiluminescence assay with luminol and horseradish peroxidase for extracellular and intracellular ROS, stimulated with serum-opsonized heat-inactivated S pneumoniae (200 bacteria/neutrophil), serum-opsonized live S aureus (5 bacteria/neutrophil), or zymosan (3 particles/neutrophil), as indicated, and quantified as area under the curve (AUC). Mock-stimulated ROS production was subtracted and is not shown for clarity. Data are mean ± standard error of the mean of the indicated numbers of independent experiments; each dot is the mean AUC from 1 experiment. Statistics comprised one-way analysis of variance with Holm-Šidák’s (S pneumoniae, S aureus) or Tukey’s (zymosan) multiple comparisons tests.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal