PMC

Mice were left untreated or received IL-1β (5ng, i.p.) and the extent of cell rolling was determined 1.5h later in A) Male (n=6-8) and female (n=6-9) eNOS^−/− mice, B) Male (n=6-7) and female (n=6-13) COX-1^−/− mice and 0-4h later in C) eNOS^−/−/COX-1^−/− (dKO, n=5-18) mice. Data are mean ± SEM. Statistical significance determined using two-way ANOVA demonstrated significant difference between the sexes of P<0.001 for panel A and B and P<0.01 (**) for panel C (in all cases interaction P value not significant) with Bonferroni post-tests shown as # for p<0.05.

Mice were left untreated or received IL-1β (5ng, i.p.) and the extent of cell rolling was determined 1.5h later in A) female wild type (WT) mice that have undergone sham (SHAM, n=7-9) or ovariectomy operation (OVX, n=5-11) and B) female OVX mice implanted with osmotic mini-pumps providing oestradiol (E, 0.4μg/day) or vehicle (VEH) control (n=8 for both). Data are mean ± SEM. Unpaired t-test for comparison of 2 groups (OVX-VEH vs OVX-E) shown as # for P<0.05 or for Bonferroni post-tests following two-way ANOVA (P<0.05) comparing sham vs OVX groups.

mRNA expression of CXCL1(KC), CXCL2(MIP2), and CXCL5(LIX) was assessed using quantitative RT-PCR of mesenteric tissue from male and female wild type mice treated with saline or IL-1β (5ng i.p., 1.5h). Data (n= 6 animals per group) are expressed as fold increase above control WT normalized to 18S. All data shown as mean ± SEM. Statistical significance determined using two-way ANOVA demonstrated no significant differences between the sexes (in all cases interaction P value not significant).

A) Representative histogram of P-selectin expression in the presence of IL-1β (20 ng/ml, 1.5h) of isotype (black) and male (red) and female (green) positive cells. B) Median fluorescence intensity (MFI) of P-selectin expression in the absence and presence of IL-1β (20 ng/ml, 1.5h). C) Representative FSC-SSC dot-plot of isolated cell population (R1). D) Representative histogram of ICAM-2 expression of positive cells (black) and isotype control (red) in R1. Data shown are mean ± SEM for 7 primary cultures of WT cells, each one prepared from 3 animals. Statistical significance determined using two-way ANOVA of *P<0.05 between the sexes (interaction P value not significant), with Bonferroni post-test shown as # for P<0.05.

Six-day old air-pouches were injected with IL-1β (20ng) at time 0, with lavage fluids being collected 4h later. A) Representative FSC/SSC dot-plot of the whole cell population collected from air pouches and histogram of Gr-1 positive expression (black line) vs. control (blue line) of gated cells in the population R1. B) Density dot-plots of air pouch lavage fluids collected from vehicle control and IL-1β (5ng) treated male wild type mice. C) Total number of Gr-1 positive cells recovered from air-pouches following vehicle or IL-1β treatment in male (n=5), female (n=5 con, n=9 IL-1 β) and ovariectomized female (OVX; n=5) mice. Data are mean ± SEM. Statistical significance using two-way ANOVA between the 3 groups P<0.05 (interaction P value not significant) with Bonferroni post-tests shown as # P<0.05.

Wild type female and male mice were treated with IL-1β (5 ng, i.p.) at time 0, and then, at the reported times, instrumented for intravital microscopy analysis of the mesenteric microcirculation conducted for assessment of A) Leukocyte rolling and B) leukocyte adhesion in female and male mice (n=10-15 animals per group). C) Typical hematoxylin and eosin (H&E) staining of mesenteric postcapillary venules (v) from a male WT mouse treated with IL-1 β (5ng, 1.5h). The arrows identify multi-lobed nuclei of granulocytes. Original magnification x400. D) Time-course of the cell recruitment in response to IL-β (5ng) as determined by measurement of myeloperoxidase (MPO) in wild type male (n=12-15) and female (n=8-15) mice. Leukocyte recruitment assessed using intravital microscopy 1.5h following administration of TNFα (300ng, i.p.) and shown as E) leukcotye rolling and F) leukocyte adhesion in male (n=8) and female (n=10) mice. All data shown as mean ± SEM of n mice per group. Statistical significance determined using two-way ANOVA shown as *P<0.05 for differences between the sexes (in all cases interaction P value not significant) with # for p<0.05 representing Bonferroni post-tests in panels A and B, Dunnetts post-test in panel D and unpaired Student’s test shown in panels E and F.