Annexin 1 (Anx-A1) is a glucocorticoid-regulated calcium-binding protein described to possess anti-inflammatory/protective properties in several experimental models of inflammation, including systemic endotoxemia) [1]. Recently, a line of mice lacking the Anx-A1 gene (Anx-A1-/-) has been generated and preliminary phenotype reported [2]. Here we have used Anx-A1-/- to test the role of this anti-inflammatory mediator in systemic inflammation and taken advantage of the construct used, which contains reporter Lac-Z gene [2], to monitor Anx-A1 gene expression during the on-going inflammatory reaction.

Male wild type (WT) and knockout (Anx-A1-/-) mice (25-30 g body weight) were treated with 10 mg/kg i.p. LPS (E. coli Serotype 0111:B4; specific activity of 500,000 U/mg). At different time-points, the whole mesentery and peritoneal cells were collected. Tissue and cell samples were fixed (4% paraformaldehyde), embedded in LR Gold resin and sectioned at 1 mm thickness for morphological studies. Cell numbers were also recorded whereas Anx-A1 gene expression was monitored by the X-Gal stain technique [2]. Finally, the survival rate of mice was also determined. Data were analysed by ANOVA followed by the Bonferroni test; survival data were analysed by the Fisher’s exact test. In all cases a P value less than 0.05 as taken as significant.

LPS treatment was associated with pronounced and restricted Anx-A1 gene expression. In the mesenteric vasculature, extravasated neutrophils were monitored (cells/cm²): 6 ±1, 25 ±2 and 15 ±2 in WT mice and 1±1, 2±1 and 5±1 in the Anx-A1-/-, as measured at 1.5 h, 6 h and 24 h post-LPS, respectively (n=5). Neutrophil recovery in the peritoneal washes was higher in Anx-A1-/- selectively at 1.5 h (10.1 ±0.6 x10⁵) respect to WT mice (1.3 ±0.1 x10⁵; n=10, P<0.05). However in WT mice there was a time-dependent increase in neutrophil recruitment which was not paralleled in Anx-A1-/- mice: for instance, at 24 h 12.4 ± 0.6 x10⁵ vs. 2.5 ± 0.5 x10⁵ cells/mouse were counted for WT and Anx-A1-/- (n=5. P<0.05). Importantly, whereas only 2 out of 20 mice died in the WT group (10%), all Anx-A1-/- mice died within 24-36 h post-LPS (P<0.05). Finally, minimal Anx-A1 gene expression was detected by X-Gal staining in endothelial cells in the absence of inflammation (4.5 ±0.3 arbitrary unit/cell). LPS treatment provoked significant increases in gene expression both in endothelial cells (55 ±5, 75 ±8 and 21 ±5 units/cell at 1.5, 6 and 24 h, respectively) as well as in extravasated neutrophils (55±5, 46 ±8 and 34 ±5 units/cell at 1.5, 6 and 24 h, respectively).

In conclusion these data indicated a dysregulated neutrophil recruitment in the absence of endogenous Anx-A1 that is accompanied with a marked systemic inflammatory response. Anx-A1-/- gene appears to be rapidly activated after LPS, not only in activated neutrophils, but also in endothelial cells. All these Anx-A1-dependent alterations are likely to be responsible for the lethal effect of LPS. Use of this new animal tool has unveiled novel characteristics for the Anx-A1 system in endotoxemia.

[1] De Coupade et al., (2001) Am J Pathol, 159, 1435-1443
[2] Hannon et al., (2003) Faseb J 17, 253-255

This work was supported by the Arthritis Research Campaign (P0583). ASD is funded by a PhD Studentship from FAPESP (02/09920-0) and a travelling studentship from CAPES (BEX0223/03-4)