Activation of the annexin A1 pathway underlies the protective effects exerted by estrogen in polymorphonuclear leukocytes.

Methods and Results Using whole blood flow cytometry, we demonstrated that pre-menopausal women expressed higher levels of surface AnxA1 on circulating PMN, compared to males. This correlated with high plasma estrogen during the menstrual cycle. The addition of estrogen in vitro to male PMN induced rapid mobilization of AnxA1, optimal at 5ng/ml and 30 min incubation period; this effect was abolished in the presence of the estrogen receptor antagonist, ICI182780. Estrogen addition to human PMN induced a distinct AnxA1 hi CD62L lo CD11b lo phenotype, and this was associated with lower cell activation as measured by microparticle formation. Treatment of human PMN with E 2 inhibited cell adhesion to an endothelial cell monolayer under shear, which was absent when endogenous AnxA1 was neutralized. Of interest, addition of estrogen to PMN flown over the endothelial monolayer amplified its upregulation of AnxA1 localization on the cell surface. Finally, in a model of intravital microscopy, estrogen inhibition of white blood cell adhesion to the post-capillary venule was absent in mice nullified for AnxA1.

Materials and Methods

Detailed information on protocols and ethics are in the Supplemental Methods file .

Cell culture and reagents

Unless otherwise stated, all cell culture reagents were obtained from Sigma-Aldrich (Poole, Dorset, UK). Human PMN were isolated via density gradient as described 17 . PMN (10 6 cells per test) or whole blood aliquots (50μl) were incubated with E 2 prior to stopping reactions on ice. In selected experiments, the estrogen receptor (ER) antagonist ICI182780 was added for 10 min prior to E 2 . In other cases, E 2 was added at 5 ng/ml (30 min) prior to stimulation with formyl-Met-Leu-Phe (fMLF; 10nM). Flow cytometric detection of surface AnxA1 on human PMN Cells or whole blood were incubated for 1 h at 4°C with mouse anti-human AnxA1 (mAb1B, produced in-house 23 or anti-human FPR2/ALX (Genovac, Freiburg, Germany), using a three-step protocol ( Supplemental Methods file ). Briefly, after the first 1-hour incubation, cells were washed and incubated with a rabbit, anti-mouse IgG FITC-conjugated. Then a further 30 min step with conjugated antibodies to CD16 (PE, clone eBioCB16) or to L-selectin (PE-Cy5,clone DREG-56) or, in some samples, to CD11b (APC, clone ICRF44) was run. For detection of plasma microparticles, after incubation with E 2 in the presence or absence of fMLF, platelet-free plasma was double stained for CD66b (PE, clone G10F5) (or IgM isotype control) to identify PMN-derived plasma microparticles and co-stained for either AnxA1, FPR2/ALX or CD62L. Beads (1-μm each; Becton Dickinson, San Jose, CA) were also run, in selected samples, to control for microparticles size. In all cases, 20,000 events were acquired by using a FACSCalibur flow cytometer (Becton Dickinson), and analysed using FlowJo analysis software (Version 9.2, Treestar Inc, Stanford, CA).

Western blot analysis

PMN were separated into cytosolic and membrane fractions as reported 24 , 25 and subjected to standard SDS–polyacrylamide gel electrophoresis (PAGE), transferred onto PVDF membranes (Millipore, Watford, United Kingdom). These were incubated with mAb1B (1 μg/ml) and HRP-conjugated goat anti–mouse IgG (Dako, Cambridge, United Kingdom). Proteins were detected using enhanced chemiluminescence (ECL) detection kit and visualized on Hyperfilm (GE Healthcare). Determination of plasma of 17-β estradiol, progesterone, and cortisol concentrations Blood was taken from females who were not on any oral contraceptives (for demographics see Supplemental Table 1 ) at days 2, 12, 19 and 26 of individual menstrual cycles. All blood samples were taken between 10 and 11 am. Plasma prepared from blood of female volunteers was tested for 17-β estradiol and progesterone concentrations using specific enzyme-immunoassays (estradiol EIA kit, Invitrogen; progesterone and cortisol EIA, Cayman Chemical Co, Inc, Ann Arbor, MI, USA) Flow chamber assay Human umbilical vein endothelial cells (HUVECs) were cultured until confluence and stimulated with 10 ng/mL tumor necrosis factor-α for 4 hours (Sigma-Aldrich). Isolated PMNs were incubated with vehicle, E 2 or E 2 plus mAb1B) for 30 min at 37°C, before flow over HUVEC monolayer at a rate of 1 dyn/cm 2 , for 8 minutes, as previously described 17 . PMN/HUVEC interaction in the flow chamber was monitored on 6 random fields recorded for 10 seconds. Analysis of total cell capture, rolling and firmly adherent PMN was carried out off-line by manual quantification. In some cases, cells flowed over the HUVEC monolayers were collected and compared with resting (pre-flowed) or adherent PMN for the extent of cell-surface AnxA1, CD62L and CD11b expression using the flow cytometric protocol described above. Intravital microscopy of leukocyte recruitment on mesenteric post-capillary venules Male C57Black6 mice (AnxA1 +/+ and AnxA1 −/− ; 4 week old) 26 were injected with either PBS or estradiol (100ng/mouse, i.p.) 30 min before administration of IL-1β (10ng/mouse, i.p.). After 2 hours, animals were anesthetized and the mesenteric microvascular bed was exposed for analysis. Intravital microscopy was performed as previously reported 18 . A cautery incision was made along the abdominal region and the mesenteric vascular bed was exteriorized, placed on a viewing Plexiglas stage, and mounted on a Zeiss Axioskop “FS” with a water-immersion objective lens (magnification 40; Carl Zeiss, Welwyn Garden City, United Kingdom) and an eyepiece (magnification x10; Carl Zeiss). Mesenteries were superfused with thermostated (37°C) bicarbonate-buffered solution at a rate of 2 mL/min. When a suitable post-capillary venule was selected (diameter 20 - 40 μm; 100 μm straight vessel length), recording was started and kept for a 3-minute period, where adherent and emigrated leukocytes were counted.

Statistical analyses

Data are expressed as mean ± SEM. Student’s t -test was used to compare two groups with parametric data distribution. For multiple comparison analyses, one- and two-way ANOVAs were carried out, with appropriate Dunnet’s, Tukey’s or Bonferroni post correction tests. P -values