Annexin 1 (ANX-A1) is a glucocorticoid-regulated 37 kDa protein shown to be particularly active in inhibiting the cellular phase of the host inflammatory response. For instance, human recombinant ANX-A1 and its peptido-mimetic Ac2-26 (derived from the N-terminus) reduced the extent of white blood cell adhesion and emigration promoted by the particulate stimulus zymosan in post-capillary venules of the mouse mesenteric microcirculation (Lim et al, 1998). The recent generation of mice deficient in ANX-A1 (Hannon et al., 2003) prompted this study with the aim of determining the putative role played by endogenous ANX-A1 on the distinct phases of the leukocyte recruitment process, as assessed by intravital microscopy.

Male ANX-A1 knockout (KO), wild type (WT) or C57/BL6 mice (25-35 g; the latter purchased from Banton and Kingsman, Hull) were anaesthetised with diazepam and Hypnorm™, and the cremaster muscle was exteriorised. One post capillary venule was chosen (length 100 µm, width 20-40 µm), superfused with a bicarbonate buffered solution (BBS) at 37°C, gassed with 5% CO₂ - 95% N₂, and monitored throughout the experiment. Red blood cell centreline velocity was measured with a Doppler velocimeter. After a 30 min stabilisation period, either BBS or platelet-activating factor (PAF; 1-100 nM in BBS) were superfused onto the microcirculation. Recordings were made every 15 min up to 90 min for later off-line analysis of the extent of leukocyte rolling (µm s^-1), adhesion (no. of static cells per 100 µm vessel wall) and emigration (no. of emigrated cells per 100x50 µm² of extravascular tissue). Data (mean ± s.e.mean) were analysed by ANOVA followed by Bonferroni's post significance test. A P value of less than 0.05 was considered significant.

In initial experiments we tested the susceptibility of the cremaster microcirculation to ANX-A1 by administering peptide Ac2-26 (200 µg) or saline (100 µl) intravenously prior to start of 100 nM PAF superfusion (time 0). The peptide significantly reduced cell emigration at the 75 min (-40%) and 90 min (-50%) time points (n=4 mice per group, P<0.05)), without affecting PAF-induced changes in cell rolling and adhesion. Experiments were then carried out to compare WT and ANX-A1 KO mice. Superfusion of the cremaster microcirculation with 100 nM PAF promoted higher extents of leukocyte rolling (max at 30 min), adhesion (from 30 min) and emigration (45 min), then BBS superfusion, however no significant differences were seen between WT and KO mice (n=6; P>0.05). When a lower concentration of PAF (1 nM) was used, significant increases in cell rolling, adhesion and emigration were again measured compared to BBS. However, this time a significantly higher degree of cell emigration was measured in ANX-A1 KO mice. Values were significant at 45 and 60 min post superfusion: WT, 4.67 ± 0.49 vs. KO, 6.67 ± 0.80 emigrated cells (n=6 mice, P<0.05), and WT, 5.00 ± 0.45 vs. KO, 7.50 ± 0.56 emigrated cells (n=6 mice, P<0.05), at 45 and 60 min, respectively. No significant changes in red blood cell velocity were measured in any of the groups analysed.

In conclusion, this study demonstrates altered leukocyte-endothelium interactions in ANX-A1 KO mice. The tonic inhibitory role exerted by this endogenous protein can be surmounted by increasing the strength of the stimulus applied.

Hannon R et al., (2003) FASEB J 17, 253-255.
Lim LHK et al., (1998) Proc Natl Acad Sci USA, 95, 14535-14539.

This work was supported by the Medical Research Council (G78/7211) and, in part, by the William Harvey Research Foundation (London, UK).