Figure 7.

A2MG preserves neutrophil responses in the presence of endotoxin: correlation of A2MG containing microparticles levels with survival in sepsis.

A  CD11b neo-epitope exposure following LPS treatment (1 μg/ml, 60 min, 37°C) of isolated human neutrophils was determined by flow-cytometry.

B  Neutrophil interaction with ICAM-1 coated chambers following incubation with LPS (1 μg/ml, 60 min, 37°C) or A2MG (10 nM, 15 min, 37°C) followed by LPS (1 μg/ml, 60 min, 37°C) under flow.

C  GRK2 expression was assessed by western blotting in human neutrophils incubated with PBS, LPS (1 μg/ml, 60 min, 37°C) or A2MG (10 nM, 5 min, 37°C) followed by LPS (1 μg/ml, 60 min, 37°C).

D  CXCR2 expression on human neutrophils incubated with LPS with or without A2MG (60 min, 37°C) as determined by flow cytometry.

E  Neutrophil chemotaxis towards IL8 (100 ng/ml) following cell incubation with LPS (1 μg/ml) and A2MG as indicated. Results are mean ± s.e.m. of n = 3–4 distinct microparticle and neutrophil preparations (*P < 0.05, **P < 0.001 versus PBS incubations, ^#P < 0.05 versus LPS; ^§P < 0.05 versus IL8+LPS by one way ANOVA).

F  Control MV; (10⁶/mouse) or A2MG-MV were administered i.v. at the indicated doses 5 min prior to CLP (see Materials and Methods for details). Mice were sacrificed at 6 h and peritoneal exudate neutrophil counts were determined by light microscopy and flow cytometry. Results are mean ± s.e.m. n = 5 mice per group (*P < 0.05 versus MV treated mice by one way ANOVA).

G  A2MG surface expression on plasma microparticles (AnxAV^positive events) from healthy volunteers (HV; n = 15), patients that survive (SS; n = 25) and those that do not survive the sepsis (SNS; n = 25) as determined by flow-cytometry (see Materials and Methods and supplementary Table S1 for further details; *P < 0.05; ***P < 0.001 versus HV, ^#P < 0.05, ^###P < 0.01 versus SS by one way ANOVA; ^§§§P < 0.01 by two tailed Fisher's exact test).