Figure 2.
Figure 2. Neutrophils from patients have large macropinocytotic vesicles, excess superoxide production and diminished fMLF-induced chemotaxis. (A) Light microscopy images of blood smears from health control (HC), patient (Pt)1, and Pt2. Large vacuoles are clearly present within the neutrophils of Pt1 and Pt2 (arrows). Images acquired from a CellaVision DM1200. Original magnification ×100. (B) Transmission electron microscopy of neutrophils from HC, Pt1, and Pt2. *Large vacuoles are seen in both patients. Paucity of specific granules in Pt1 compared with HC. Images were acquired using a Hitachi H7600 transmission electron microscope and an AMT XR41B digital camera. Original magnification of HC and Pt2, ×3000; original magnification of Pt1, ×2500. (C) Neutrophils from HC either fresh (left) or shipped along with the patient sample (middle) and Pt2 (right) were incubated with FITC-dextran for 15 minutes. Cells were fixed and viewed using Images and were acquired using EVOS FL system with ×40 and ×100 magnification and analyzed with ImageJ software to determine the cell area, integrated density, and mean. Corrected total cell fluorescence = integrated density – (area × mean of background readings). (D) Total fluorescence of FITC-dextran cells corrected for background fluorescence. Calculations from 10 to 30 individual cells from each sample were plotted. P values were calculated using 1-way ANOVA correcting for multiple comparisons. ****P < .0001. (E) Neutrophil extracellular superoxide production kinetics measured every 15 seconds after fMLF treatment of cells from Pt1 (red) and Pt2 (green) compared with 6 HCs (blue line with error bars) showing superoxide production over 30 minutes, evaluated every 15 seconds. (F) Cumulative superoxide production after 30 minutes. (G) Net chemotactic velocity of 10 individual cells from patients 1 and 2 compared with 2 HC with buffer alone (left) or fMLF (right). Two-way analysis of variance (ANOVA) correcting for multiple comparisons was used for calculating significance. ****P < .0001. (H) Individual cell tracings from Pt1 (right) and HC (left) tracking movement and colored by 10-minute increments. AU, arbitrary unit; O.D., optical density.

Neutrophils from patients have large macropinocytotic vesicles, excess superoxide production and diminished fMLF-induced chemotaxis. (A) Light microscopy images of blood smears from health control (HC), patient (Pt)1, and Pt2. Large vacuoles are clearly present within the neutrophils of Pt1 and Pt2 (arrows). Images acquired from a CellaVision DM1200. Original magnification ×100. (B) Transmission electron microscopy of neutrophils from HC, Pt1, and Pt2. *Large vacuoles are seen in both patients. Paucity of specific granules in Pt1 compared with HC. Images were acquired using a Hitachi H7600 transmission electron microscope and an AMT XR41B digital camera. Original magnification of HC and Pt2, ×3000; original magnification of Pt1, ×2500. (C) Neutrophils from HC either fresh (left) or shipped along with the patient sample (middle) and Pt2 (right) were incubated with FITC-dextran for 15 minutes. Cells were fixed and viewed using Images and were acquired using EVOS FL system with ×40 and ×100 magnification and analyzed with ImageJ software to determine the cell area, integrated density, and mean. Corrected total cell fluorescence = integrated density – (area × mean of background readings). (D) Total fluorescence of FITC-dextran cells corrected for background fluorescence. Calculations from 10 to 30 individual cells from each sample were plotted. P values were calculated using 1-way ANOVA correcting for multiple comparisons. ****P < .0001. (E) Neutrophil extracellular superoxide production kinetics measured every 15 seconds after fMLF treatment of cells from Pt1 (red) and Pt2 (green) compared with 6 HCs (blue line with error bars) showing superoxide production over 30 minutes, evaluated every 15 seconds. (F) Cumulative superoxide production after 30 minutes. (G) Net chemotactic velocity of 10 individual cells from patients 1 and 2 compared with 2 HC with buffer alone (left) or fMLF (right). Two-way analysis of variance (ANOVA) correcting for multiple comparisons was used for calculating significance. ****P < .0001. (H) Individual cell tracings from Pt1 (right) and HC (left) tracking movement and colored by 10-minute increments. AU, arbitrary unit; O.D., optical density.

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