Figure 7.
Figure 7. Neutrophil sequestration by MZ macrophages contributes to TI antibody response after pneumococcal infection. (a) Representative confocal images and quantification of neutrophil (green) colocalization with MZ macrophages (MZM; red) and RP macrophages (RPM; purple) after saline (black bars) or S. pneumoniae (S. p.; 24 h, blue bars; 48 h, red bars) i.v. injection. Bars, 50 µm. Eight fields of view were averaged per animal. n = 4–6 pooled from four independent experiments. (b) Time-lapse two-photon microscopy images of neutrophil behavior in MZ 24 h after S. pneumoniae injection. Green, neutrophils; red, MZM; multicolored, tracks. Bars, 25 µm. (c–e) Tracking quantification of neutrophil cell velocity (c), dwell time (d), and MZ macrophage interaction time (e) at 24 h (blue bars) and 48 h (red bars) after S. pneumoniae i.v. infection. Two fields of view were averaged per animal. n = 3–4 pooled from four independent experiments. (f) Quantification of neutrophil colocalization with MZ B cells and RP macrophages at 24 h after S. pneumoniae i.v. infection in control (Ctrl) liposomes and low-dose CLL–treated animals. n = 3–5 pooled from three independent experiments. (g) Quantification of neutrophil colocalization with MZ B cells (left) and RP macrophages (right) at 24 h after S. pneumoniae i.v. infection in animals treated with isotope control, anti-CD18, anti-CD29, and anti–VCAM-1 blocking antibodies. Eight fields of view were averaged per animal. n = 3–5 pooled from three independent experiments. (h) Representative image of neutrophil localization within the MZ B cells (MZB) 24 h after i.v. S. pneumoniae infection. Green, MZ B cells; red, neutrophils; purple, RP macrophages. Data are representative of n = 4 from two independent experiments. Bar, 25 µm. (i) Quantification of S. pneumoniae–specific serum IgM and IgG antibody levels in isotype antibody–treated and Ly6G (1A8) antibody (1A8 Ab)–treated animals at 72 h after infection. Red dotted lines indicate antibody levels in MZ B cell–depleted animals. n = 5–9 total pooled from three independent experiments. (j) S. pneumoniae bacterial counts in the blood and spleen at 72 h after i.v. infection in isotype antibody–treated and Ly6G (1A8) antibody–treated animals. Black lines show the median, and red dotted lines show the detection limit. n = 5–9 total pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANOVA (a and e), two-way ANOVA (c and d), Student’s t test (f, g, and i), and Mann-Whitney test (j) statistical analyses were performed.

Neutrophil sequestration by MZ macrophages contributes to TI antibody response after pneumococcal infection. (a) Representative confocal images and quantification of neutrophil (green) colocalization with MZ macrophages (MZM; red) and RP macrophages (RPM; purple) after saline (black bars) or S. pneumoniae (S. p.; 24 h, blue bars; 48 h, red bars) i.v. injection. Bars, 50 µm. Eight fields of view were averaged per animal. n = 4–6 pooled from four independent experiments. (b) Time-lapse two-photon microscopy images of neutrophil behavior in MZ 24 h after S. pneumoniae injection. Green, neutrophils; red, MZM; multicolored, tracks. Bars, 25 µm. (c–e) Tracking quantification of neutrophil cell velocity (c), dwell time (d), and MZ macrophage interaction time (e) at 24 h (blue bars) and 48 h (red bars) after S. pneumoniae i.v. infection. Two fields of view were averaged per animal. n = 3–4 pooled from four independent experiments. (f) Quantification of neutrophil colocalization with MZ B cells and RP macrophages at 24 h after S. pneumoniae i.v. infection in control (Ctrl) liposomes and low-dose CLL–treated animals. n = 3–5 pooled from three independent experiments. (g) Quantification of neutrophil colocalization with MZ B cells (left) and RP macrophages (right) at 24 h after S. pneumoniae i.v. infection in animals treated with isotope control, anti-CD18, anti-CD29, and anti–VCAM-1 blocking antibodies. Eight fields of view were averaged per animal. n = 3–5 pooled from three independent experiments. (h) Representative image of neutrophil localization within the MZ B cells (MZB) 24 h after i.v. S. pneumoniae infection. Green, MZ B cells; red, neutrophils; purple, RP macrophages. Data are representative of n = 4 from two independent experiments. Bar, 25 µm. (i) Quantification of S. pneumoniae–specific serum IgM and IgG antibody levels in isotype antibody–treated and Ly6G (1A8) antibody (1A8 Ab)–treated animals at 72 h after infection. Red dotted lines indicate antibody levels in MZ B cell–depleted animals. n = 5–9 total pooled from three independent experiments. (j) S. pneumoniae bacterial counts in the blood and spleen at 72 h after i.v. infection in isotype antibody–treated and Ly6G (1A8) antibody–treated animals. Black lines show the median, and red dotted lines show the detection limit. n = 5–9 total pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANOVA (a and e), two-way ANOVA (c and d), Student’s t test (f, g, and i), and Mann-Whitney test (j) statistical analyses were performed.

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