Figure 4. B cells impact neutrophils, the key effectors driving sepsis-induced tissue damage.

(A) Aspartate aminotransferase (ASAT) plasma levels 18 hr p.i. with E. coli in lipopolysaccharide (LPS) or NaCl pretreated mice, in which platelets, monocytes/macrophages, or neutrophils, respectively, were depleted before infection. (B) Flow-cytometric analysis of bone marrow neutrophils and B cells after i.v. administration of LPS at time = 0 hr. (C) Flow-cytometric analysis of neutrophils in the bone marrow of wildtype, Rag2^-/- and J_HT mice 2 weeks after LPS or NaCl treatment. (D–E) Flow-cytometric analysis of neutrophils of wildtype mice pre-treated with NaCl or LPS, respectively, and infected for 18 hr with E. coli, in blood (D) and peritoneal lavage fluid (PLF) (E). (F–G) Quantification of (F) immunohistological staining for NIMP-R1⁺ cells on liver sections (G) of mice pretreated with NaCl or LPS, respectively, and infected with E. coli for 18 hr. (H–J) Flow-cytometric analysis of neutrophils 18 hr p.i. with E. coli in blood (H), PLF (I), and liver (J) of J_HT mice. (K) Flow-cytometric analysis of blood neutrophil CD62L expression of WT, Rag2^-/- and J_HT mice at 18 hr p.i. with E. coli. Data in (A) shown for the control group and neutrophil depletion are pooled from two independent experiments (n=4–6/experimental group), platelet and monocyte/ macrophage depletion represent a single experiment (n=8/group). Data in (B), (D–E), (F), and (J) are pooled from two independent experiments (n=4–8/experimental group). Data in (H–I) are representative of two experiments (n=5–8/group). Data in (K) are from a single experiment (n=4–8/group). All data are presented as mean +/-SEM. * p≤0.05, ** p≤0.01 and *** p≤0.001.

Figure 4—figure supplement 1. B cells impact neutrophils, the key effectors driving sepsis-induced tissue damage.

(A) Peritoneal lavage fluid (PLF) cell numbers of uninfected mice and mice 18 hr p.i. with E. coli, which received an anti-Ly-6G depletion antibody i.v. 24 hr before infection. (B) Flow-cytometric analysis of blood platelets (SSCA_low, CD61⁺) in mice that received an anti-GPIbα (R300) depletion antibody 24 hr earlier. (C) Immunohistological staining for F4/80⁺ cells on liver sections of mice which received either clodronate-loaded liposomes (clodrolip) or empty control liposomes i.v. 24 hr earlier. (D) E. coli CFUs 18 hr p.i. with E. coli in livers of NaCl or lipopolysaccharide (LPS) pretreated mice, which received depletion antibodies for platelets or neutrophils, or clodronate liposomes before infection. (E) Flow-cytometric analysis of bone marrow neutrophil abundance 2 weeks after treatment with LPS or NaCl. (F) Quantification of NIMP-R1⁺ cells in immunohistological staining of lung sections from NaCl or LPS pretreated mice 18 hr p.i. with E. coli. (G–H) Flow-cytometric analysis of neutrophils in blood and PLF of NaCl or LPS pretreated Rag2^-/- mice 18 hr p.i. with E. coli. (I) Quantification of NIMP-R1⁺ cells after immunohistological staining of liver sections from NaCl or LPS pretreated Rag2^-/- mice 18 hr p.i. with E. coli. Data shown in (A), (E) and (H) are representative out of two independent experiments (n=2–6 per experimental group for A and E and n=7–8 per experimental group for H). Data in (B–C) are from a single experiment, which was set up to test the depletion efficiency in uninfected animals (n=3 per experimental group). In (D) CFU data shown for the control group and neutrophil depletion are pooled from two independent experiments (n=4–6 per experimental group) and data showing the platelet and monocyte/macrophage depletion are from a single experiment (n=8 per group). Data depicted in (G) and (I) are pooled from two independent experiments (n=4–6 per experimental group) and data in (F) are from a single experiment (n=8 per group). All data are presented as mean +/-SEM. * p≤0.05.