Suppression of endothelial P-selectin expression contributes to reduced cell trafficking in females: an effect independent of NO and PGI₂

Abstract

Objectives

Sex hormones underlie the lower incidence of cardiovascular disease in premenopausal women. Vascular inflammation is involved in the pathogenesis of several cardiovascular diseases and it has been reported that sex hormones modulate inflammatory responses but mechanisms responsible for these effects are not yet fully established. Herein, we assessed whether sex differences in leukocyte recruitment might exist and investigated the underlying mechanisms involved in this response.

Methods and Results

IL-1β or TNFα treatment caused leukocyte rolling, adhesion and emigration in mesenteric postcapillary venules in vivo that was substantially reduced in female mice compared to male mice; this difference was abolished by ovariectomy and partially restored by oestrogen replacement. Deletion of endothelial nitric oxide synthase (eNOS) or cyclooxygenase-1 (COX-1) alone, or in combination, did not alter the leukocyte recruitment in IL-1β-treated females, but significantly enhanced this response in male mice. Treatment of murine pulmonary endothelial cells with IL-1β increased expression of P-selectin in male but not female cells.

Conclusion

Thus, we demonstrate a profound oestrogen-dependent and NO and PGI₂-independent suppression of leukocyte recruitment in females.

Introduction

A lower incidence of cardiovascular disease (CVD) in pre-menopausal women compared to age-matched male counterparts and post-menopausal women^1;2 suggests that female sex underlies a protective effect on the cardiovascular system. Indeed, a wealth of evidence from observational and experimental studies, in both animals and humans, supports the concept of a protective effect of ovarian hormones, predominantly oestrogens^3;4. Understanding the molecular pathways that underlie the beneficial effects of oestrogens may, therefore, identify novel strategies that could be taken to harness the therapeutic potential of hormone therapy safely and novel targets to treat CVD in both sexes.

A number of targets/pathways/mediators have been proposed to play a role in mediating the beneficial effects of female sex hormones^3;5-7. Of these mechanisms there is a substantial body of evidence implicating oestrogen-induced or enhanced activation of the endothelium⁵. Endothelial dysfunction is thought to be instrumental in precipitating the vascular inflammation that is an early and crucial event in the pathogenesis of a number of cardiovascular disorders (e.g. atherosclerosis, ischaemia/reperfusion [I/R] injury)^8;9. In health, the endothelium is critical in maintaining an anti-inflammatory, and thereby anti-atherogenic, phenotype of the blood vessel wall^10;11. This activity has been attributed to its capacity to release factors that not only alter the tone and growth of the underlying smooth muscle, but also regulate the reactivity of circulating white cells, erythrocytes and platelets, and govern vascular permeability. The prevailing wisdom currently promotes the thesis that oestrogens upregulate the synthesis, release and activity of protective endothelial factors and, thereby, sustain the protective phenotype conveyed by the endothelium and underpin the protection of females from CVD⁷. A significant proportion of this cytoprotective activity of the endothelium has been attributed to alterations in NO bioavailability either by upregulation of synthesis and/or activity^5;12. However, the role of other potentially beneficial endothelial mediators including prostacyclin (PGI₂) and endothelium-derived hyperpolarising factor (EDHF) has received little attention. There is some evidence, from diverse animal models of inflammation, to suggest the existence of sex hormone dependent male/female differences in inflammatory cell recruitment^13-18. However, the exact pathways i.e. whether this regulation occurs at the level of the endothelium or at the level of the circulating cell and the mediators involved are uncertain.

Herein, we demonstrate that reduced expression of P-selectin underlies the attenuated leukocyte recruitment that occurs in female mice in response to acute inflammatory stimuli. Moreover, we demonstrate that whilst both NO and PGI₂ tonically repress leukocyte recruitment in response to inflammatory stimuli in males neither mediator has a role in females.

MATERIAL AND METHODS

Animals

All experiments were conducted according to the Animals (Scientific Procedures) Act 1986 (United Kingdom). The experiments were performed on age-matched (6-7 weeks) female and male wild-type (WT; C57BL/6, Charles River, UK), eNOS knockout (eNOS^−/−)¹⁹, COX-1 knockout (COX-1^−/−)²⁰, eNOS^−/−/COX-1^−/− double knockout (dKO) mice²¹. All knockout mice were bred in-house. In addition female WT ovariectomized (OVX) and sham-operated control (SHAM) animals were purchased from Charles River, UK. Ovariectomy was performed on sexually immature mice at 4 weeks of age. In 16 OVX mice Alzet mini-osmotic pumps (Model 1004) were implanted at the time of surgery (Charles River, UK), containing vehicle (polyethylene glycol 400; Sigma, UK) or a replacement dose of oestrogen of 0.4μg/day providing ‘physiological’ levels in mice as previously demonstrated²². Experiments were conducted on these animals 2 weeks after surgery. Mice were maintained in a 12-hr light/dark cycle room with free access to food and water.

Plasma [oestrogen] determination

Blood was collected into heparin by cardiac puncture from mice anaesthetised with isoflurane. Following centrifugation at 14,000 g for 10 min at 4°C plasma was collected and oestrogen concentration determined according to the manufacturer’s instructions using a commercially available enzyme-linked immunoassay (estradiol EIA, Cayman Chemical Co, Inc).

Myeloperoxidase (MPO) activity

Mesenteric tissue, collected at time 0, 90 min or 4 h after IL-1β (5ng/mouse, i.p.) treatment, was homogenized in 1 ml of 0.5% hexadecyltrimethylammonium bromide in MOPS buffer (10 mM, pH 7). After homogenization, samples were centrifuged at 4000 x g for 20 min at 4°C and the supernatant was collected for determination of MPO levels as previously described²³. MPO levels are expressed relative to protein content as determined by Bradford assay.

Hematoxylin and eosin staining

Whole sections of mesentery were prepared on microscope slides and dried overnight. General structures were identified by staining with hematoxylin and eosin (H&E) to enable identification of polymorphonuclear (PMN) and mononuclear cells.

Intravital microscopy

WT, eNOS^−/−, COX-1^−/− and dKO (10-15 g) mice received either murine IL-1β (5 ng; PeproTech, UK)²⁴, murine TNFα (300ng, R & D Systems, UK)²⁵ or saline vehicle i.p. After 1.5, 4 or 24 h mice were anesthetized with diazepam (6 mg/kg, s.c.) and Hypnorm (0.7mg/kg fentanyl citrate and 20 mg/kg fluanisone, i.m.), cautery incisions were made along the abdominal region, and the mesenteric vascular bed was exteriorized for intravital microscopy recordings (see online methods supplement for full details). The extent of basal and cytokine-elicited leukocyte rolling was analyzed by counting the number of cells passing a fixed point per minute (cells/min). A leukocyte was considered to be adherent to venular endothelium if it remained stationary for a period equal to or greater than 30 s. Adherent cells were expressed as the number per 100 μm length of venule counted over 1 min.

Air pouch model

IL-1β was used to induce cell migration (>90 % neutrophils) into the mouse air-pouch as previously described²⁶. On day 6, 20 ng murine recombinant IL-1β was dissolved in 0.5 ml of 0.5 % carboxymethylcellulose (CMC) and injected into the pouches. Control mice received CMC alone. In both cases air pouches were washed 4 h after administration of the stimulus with 2 ml of phosphate buffered saline (PBS) containing heparin (50 U/ml) and EDTA (3 mM) and the samples collected. These were then centrifuged at 220 g for 15 min at 4°C and pellets resuspended in 2 ml of PBS containing heparin and EDTA. PMN numbers were estimated by counting after specific staining with Turk’s solution using a Neubauer haematocytometer.

Murine Primary Lung endothelial cell culture

Endothelial cells were prepared from lungs of C57BL6 wild type mice according to validated and standard techniques^27;28 (see online methods supplement for details).

Flow Cytometry

Whole blood, air pouch supernatants and murine endothelial cell cultures were collected and subjected to FACs analysis to assess both cell types and adhesion molecules expressed using a FACScalibur flow cytometer (Becton Dickinson, CA) using CellQuest™ software (Becton Dickinson, CA) (see online methods supplement for full details).

Real-time quantitative RT-PCR

Expression of key neutrophil chemokine mRNA (CXCL1, CXCL2, CXCL5) in mouse mesenteric tissue was determined by real-time quantitative RT-PCR (see online methods supplement for details).

Statistical Analysis

All data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 5.0 (San Diego, US) and significance determined using Student’s t test for differences between two data groups, one-way ANOVA for more than two groups and two-way ANOVA for comparison between sexes with and without cytokine treatment followed by Bonferroni post-test where appropriate. The n values quoted similarly indicate the number of experiments and animals used.

RESULTS

Sex differences in granulocyte infiltration in mouse air-pouch model

IL-1β caused a significant increase in GR-1 positive (Figure 1A,B) leukocyte recruitment into air-pouches of male but not female WT mice, as assessed at the 4h time-point (Figure 1C). In contrast to intact female mice, cell recruitment was significantly raised in response to IL-1β in OVX mice (Figure 1B).

Figure 1. Flow cytometry analysis of leukocytes infiltrated into air-pouches.

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.

Plasma [oestrogen] was substantially lower in OVX (28.1 ±8.5 pg/mL, n=12) compared to SHAM mice (46.01± 6.4 pg/mL, n=16; p<0.05). The remaining measured oestrogen likely relates to extra-gonadal generation due to the activity of aromatase²⁹. In addition uterus weight was significantly decreased in OVX mice (9.4 ± 5 mg, n=22) compared to SHAM mice (82.1 ±16.5 mg, n=21, p<0.001). There were no differences in body weight between groups (OVX=21.09±0.8 g; SHAM=20.7±0.5 g). These results confirm successful surgery in OVX mice.

Leukocyte rolling studies

Sex differences in leukocyte rolling

IL-1β (5ng, i.p.) treatment caused a time-dependent increase of leukocyte rolling and adhesion that was significantly greater in magnitude in male compared to female mice (Two-Way Anova P<0.05; Figure 2A and 2B). Histological assessment of whole mesentery demonstrated that neutrophils represented the predominant cell type recruited in these experimental conditions (Figure 2C). Accordingly, in a separate experiment a time-dependent increase in MPO levels was evident from 0-4 h following IL-1β treatment in male but not female mice (Two Way ANOVA P<0.05; Figure 2D). Since the greatest difference in cell rolling between the sexes was evident at 1.5 h following cytokine treatment (26.9±7.9 cell/min [n=14] in male and 9.7±1.3 [n=9] cell/min in female, Figure 2A), this time-point was used to investigate the mechanisms involved in the sex differences in further experiments. This sex difference in leukocyte recruitment was also apparent in response to the distinct cytokine TNFα (Figure 2E and F), where both leukocyte rolling and adhesion were significantly lower in female (n=8) compared to male (n=10) mice. There were no significant differences in venular hemodynamics between the sexes (Table 1).

Figure 2. Leukocyte-endothelial cell interactions in mouse mesenteric postcapillary venules.

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.

Table 1.

Hemodynamic parameters in post-capillary venules of wild type (WT), eNOS^−/−, COX^−/− and double KO (dKO) mice under basal conditions.

Mouse Sex/Genotype	Venule diameter (μm)	Centre line velocity (mm/s)	Wall shear (s−1)
Male WT	25.6 ± 1.0	2.4 ± 0.4	486 ± 66.5
Female WT	24.0 ±0.9	1.8 ± 0.1	386 ± 33.02
Male eNOS−/−	26.0 ±0.8	2.2 ± 0.3	450 ± 44.0
Female eNOS−/−	27.5 ±3.2	2.6 ± 0.7	539 ±115
Male COX-1−/−	26.6 ± 1.9	1.8 ± 2.2	370 ± 29.3
Female COX-1−/−	27.0 ±2.0	1.4 ± 0.03	304.3 ± 28.7
Male dKO	22.3 ±0.3	1.3 ± 0.1	294.7 ± 32.9
Female dKO	29.8 ±2.6	1.3 ± 0.1	249.2 ± 41.0

Data are mean ± SEM of 4 animals per group. No significant differences detected between the sexes for each genotype as determined with unpaired t-tests.

Role of sex hormones in sex differences on leukocyte rolling

Whilst ovariectomy had no effect on basal leukocyte rolling compared to sham-operated animals, IL-1β-induced leukocyte rolling was significantly increased in OVX mice (Two-Way ANOVA P<0.01; Figure 3A). This effect of ovariectomy on the response to IL-1β was in part reversed by oestrogen replacement (Figure 3B, n=8 for both groups, P<0.05).

Figure 3. Ovariectomy alters basal and IL-1β-stimulated leukocyte rolling in mouse mesenteric post-capillary venules.

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.

IL-1β elevates chemokine expression in both sexes

Quantitative PCR of mesenteric tissue of IL-1β-treated WT animals revealed increases in mRNA expression, above that measured in saline-treated controls (Figure 4), of all three of the neutrophil-specific chemokines measured. However, this IL-1β-induced chemokine elevation was similar in both sexes (Two Way ANOVA-not significant; Figure 4).

Figure 4. Chemokine gene product levels in mesenteric tissues.

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).

Effect of IL-1β on circulating blood cells

IL-1β (5ng, i.p.) provoked a significant rise in the number of circulating granulocytes in male compared to female WT mice. No significant differences in circulating monocyte or lymphocyte numbers were evident between the sexes (Table 2). FACS analysis demonstrated PSGL-1 on the surface of all cell types measured in both sexes. Additionally L-selectin expression was significantly elevated by cytokine treatment on granulocytes in both sexes. Interestingly, however, the percentage of granulocytes expressing L-selectin under basal conditions was higher in male compared with female mice (Table 3).

Table 2.

Total number of leukocytes in whole blood of female and male wild type mice under basal conditions and after IL-1β stimulation.

Cell type	Female control	Male control	Female + IL-1β	Male + IL1β
Granulocytes	1.954 ±0.8	1.2 ±0.4	2.3 ±1.0	3.79 ±1.4*
Lymphocytes	7.04 ±0.75	6.55 ±0.91	6.46 ±0.76	6.56 ± 1.17
Monocytes	1.96 ±0.52	1.63± 0.74	2.98 ±0.22	2.87 ±0.58

Mice were left untreated or received IL-1β (5ng i.p., 1.5h). Data (number of cells × 10⁵/ml) shown are mean ± SEM for 5 animals per group. Statistical analysis using one way ANOVA followed by Bonferroni post-tests shown as *P<0.05 for control vs. IL-1β.

Table 3.

L-selectin and PSGL-1 expression in circulating leukocytes in basal conditions and after IL-1β stimulation.

Cell type (adhesion molecule)	Female control	Male control	Female + IL-1β	Male +IL1β
Granulocytes (L-selectin)	26.8 ± 1.7	39.4± 3.6	51.8±6.7	54.0±5.5
Granulocytes (PSGL-1)	99.9± 0.1	99.6± 0.1	99.6± 0.07	99.8± 0.07
Lymphocytes (L-selectin)	46.6± 4.1	44.9± 5.9	51.6± 7.5	50.8± 9.2
Lymphocytes (PSGL-1)	98.8± 0.1	97.1± 1.9	99.9± 0.03	99.8± 0.05
Monocytes (L-selectin)	31.9± 10.6	20.9± 1.2	45.8± 7.7	42.1± 6.4
Monocytes (PSGL-1)	98.4± 0.3	97.3± 1.0	99.1± 0.2	99.0± 0.2

Values report the percentage of L-selectin positive and PSGL-1 positive, events for each respective leukocyte population. Mice were left untreated or received IL-1β (5ng, i.p., 1.5h). Data are shown as mean ± SEM for 3-5 animals per group.

Sex differences in P-selectin expression on primary murine endothelial cells

Treatment with IL-1 β (20ng/ml; 1.5h treatment) did not stimulate P-selectin expression (Figure 5A-B) in cultures of female primary endothelial cells (94.1 ± 0.5% purity as identified by positive ICAM-2 expression, Figure 5C-D), in contrast P-selectin expression was significantly elevated in corresponding male endothelial cells (Two Way ANOVA P<0.05: Fig 5B).

Figure 5. Flow cytometry analysis of murine primary endothelial cells.

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.

Sex differences in basal and IL-1β –induced leukocyte rolling: role of endothelial NOS and COX-1 enzymes

Basal leukocyte rolling was significantly greater in male compared to female eNOS^−/− mice (Two-Way ANOVA P<0.001; Figure 6A) and whilst IL-1β treatment appeared to cause a minor elevation of leukocyte rolling in both sexes this did not reach significance (Figure 6A). Basal leukocyte rolling in female COX-1^−/− mice was similar to male COX-1^−/− mice (Figure 6B), however, whilst IL-1β caused a profound increase in rolling in males no such effect was evident in age-matched female COX-1^−/− animals (Two Way ANOVA P<0.001; Figure 6B). These effects were, again, unrelated to differences in venular hemodynamics which did not vary between the groups (Table 1).

Figure 6. Basal and IL-1β-stimulated leukocyte rolling in mouse mesenteric post-capillary venules.

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.

Sex differences in IL-1β –induced leukocyte rolling of dKO mice

IL-1β treatment significantly increased leukocyte rolling in male dKO mice whilst in female dKO rolling remained at basal levels up to 4h post cytokine treatment (Figure 6C). Venular hemodynamics were not altered in either genotype or sex in comparison to WT controls (Table 1).

DISCUSSION

Female sex exerts a permissive influence over inflammatory responses; a phenomenon thought to be driven principally by the activity of female sex hormones and believed to play an important role in underlying the cardioprotection evident in pre-menopausal females^3;4. However, the exact mechanisms involved in this effect are unclear. In this report we have demonstrated the existence of a sex-difference in leukocyte recruitment under inflammatory conditions and implicated female sex hormones in mediating this effect. In addition we have demonstrated that neither NO or PGI₂ is involved in this effect and propose that EDHF is likely to play a crucial role in mediating this protective phenotype. In addition, our findings suggest that at least one of the potential downstream target(s) for these beneficial effects is P-selectin, a key endothelial cell adhesion molecule involved in the early stages of the inflammatory cell recruitment process³⁰.

Several studies have demonstrated that sex hormones (particularly oestrogen) suppress leukocyte recruitment evident in innate immune responses in experimental models of inflammation using both in vivo and in vitro models^13;14;16-18. In accord with these studies we observed that leukocyte-recruitment into murine air-pouches following treatment with the cytokine IL-1β was reduced in female mice compared to age-matched male WT mice. This effect is likely to reflect a generalised decrease in reactivity of the female microvasculature since the responses to the distinct pro-inflammatory cytokine, TNFα, were also suppressed in female compared to male mice. It is possible, however, that these suppressive effects relate specifically to cytokine-induced cell recruitment since it has been recently demonstrated that female sex worsens leukocyte recruitment in response to an ischaemia/reperfusion insult in the hepatic microcirculation, albeit of rats³¹.

Our findings also suggest that female sex hormones play a role in mediating this apparent reduced sensitivity to cytokines since ovariectomy of mice resulted in a significant increase in cell recruitment in response to IL-1β that was similar in magnitude to the response evident in males. Previous characterisation of this model of inflammation indicates that the cellular infiltrate in response to IL-1β at this time-point (4h) is predominantly neutrophilic³²; a view in part supported in the present study by FACS analysis indicating that the cells recruited were granulocytes. Interestingly, whilst substantial changes in cell number were evident in response to IL-1β, there were no significant differences in the number of cells recruited in response to the vehicle control, suggesting that sex hormones play an important role in controlling leukocyte recruitment principally under inflammatory conditions. There is considerable evidence suggesting that the beneficial effects of female sex hormones relate predominantly to the activity of oestrogen^7;33, and to determine whether this hormone may underlie the effects of female sex in the present study we investigated the effects of oestrogen replacement in OVX females. Our data suggests that the sex differences are, in part, likely to be related to a suppressive effect of oestrogen, since the effects of ovariectomy were partially inhibited by oestrogen replacement. The dose of oestrogen used in our studies to ‘replace’ the levels of this hormone in female mice has previously been shown to restore physiological levels and to exert anti-inflammatory effects in mouse models²². The incomplete reversal of the effect of the cytokine may indicate that other non-oestrogenic influences/factors also play a role, such as progesterone.

Leukocyte recruitment to sites of tissue injury is a dynamic multistep process involving leukocyte rolling, adhesion, and emigration³⁰. To investigate whether the altered cell recruitment evident in the air-pouch studies might be due specifically to a sex-dependent suppression of a specific step in the recruitment paradigm we used the technique of intravital microscopy. Our findings demonstrate that enhanced leukocyte rolling and adhesion induced by IL-1β are suppressed in female WT mice compared to male mice; an effect that was not due to inherent differences in venular hemodynamics. Histological analysis of the mesenteric vasculature confirmed that the cellular target for this effect was the neutrophil, a fact substantiated by measurement of MPO in whole mesentery homogenates.

We propose that our data supports the thesis that oestrogens effect tight control on the early process of cell recruitment (leukocyte rolling phenomenon), rather than a generalised effect upon all steps of the cell recruitment paradigm i.e. rolling, adhesion and emigration. This is supported by the fact that whilst IL-1β treatment resulted in an increase in the levels of neutrophil chemokines that we have previously shown to be pivotal in IL-1β-induced cell adhesion (namely CXCL1, 2 and 5)³⁴, no difference between the levels of expression were evident between the sexes. These findings are congruent with observations in healthy volunteers demonstrating no difference in serum levels of CXCL1³⁵, and other CXC chemokines³⁶ .

Our findings, and proposed model, are at odds with studies suggesting higher levels of certain CXCL chemokines in the sera of healthy women versus age-matched men³⁷ at baseline or following an inflammatory stimulus of lipopolysaccharide³⁶, and studies demonstrating estrogen-induced suppression of rat vascular CXCL chemokine expression in response to the inflammatory stress of vascular balloon injury in vivo³⁸ or expression by cultured smooth muscle cells in response to TNFα³⁹. The reason for this discrepancy is uncertain but may reflect differences in the inflammatory stimulus or the stage at which the inflammatory response was sampled following initiation of the response.

We considered whether the differences in cell recruitment might simply reflect differences in circulating cell numbers. Indeed, our studies demonstrated that whilst under control conditions there were no significant differences in the circulating numbers of granulocytes, monocytes or lymphocytes between sexes, following IL-1β treatment granulocyte numbers rose significantly in male but not female mice. Supporting these findings are observations that the risk of coronary artery disease is correlated to increasing circulating granulocyte numbers⁴⁰ and particularly MPO levels (implicating neutrophils⁴¹) in men; an effect that is not evident in women. It is unlikely that the differences in the extent of circulating cell might relate to specific sex-differences in the levels of leukocyte adhesion molecule expression since no differences in either L-selectin or PSGL-1, the key leukocyte adhesion molecules involved in neutrophil rolling³⁰, were found in our study. Thus, it is likely that the lower numbers of circulating cells, in part, underlies the lower numbers recruited in response to IL-1β. However, sex-differences in the numbers of cells recruited (air-pouch experiments) in response to IL-1β persists even after normalisation for circulating cell numbers giving values of near 1 × 10⁵ cells for females and 8 ×10⁵ cells for males. This observation suggests that sex-differences in cell recruitment pathways likely play a role.

Several studies have demonstrated that the beneficial effects of female sex hormones within the circulation relate to upregulation of pathways at the endothelial cell critical in maintaining an anti-inflammatory phenotype of the blood vessel wall^7;42;43. Evidence supports the view that oestrogens upregulate the synthesis, release, and activity of protective endothelial factors and suppress the expression of pathogenic mediators by the endothelium^5;42. NO and PGI₂ have been identified as important targets for oestrogen activity³, however, our investigations using mice with targeted disruption of either eNOS, COX-1, or both, the principal enzymes involved in endothelial generation of NO and PGI₂ respectively, demonstrated that neither of these endothelial mediators played a role in mediating the protection against inflammatory cell recruitment in females. In contrast, the loss of either or both of these proteins resulted in substantial increases in leukocyte rolling under basal conditions and following cytokine treatment in male mice. These findings suggest that whilst both NO and PGI₂ play important roles in sustaining the anti-inflammatory phenotype of endothelial cells in male mice, they are redundant in female animals. This profile of reactivity with respect to inflammatory cell recruitment mimics our findings with respect to other differences evident in the vasoprotective effects of endothelial mediators between the sexes. Recently, we proposed that whilst NO and PGI₂ play essential roles in maintaining a vasodilated microvasculature of male mice, it is predominantly EDHF that subserves this role in female mice. Moreover, that this provision of EDHF is the predominant endothelial-derived vasorelaxant factor that confers protection against hypertension in females²¹. It is possible, therefore, that the differences between the sexes demonstrated in the present study reflect a difference in EDHF bioactivity, and support the view that the remit of EDHF extends beyond vasodilatation.

The identity of EDHF remains a hotly disputed issue, with a number of distinct candidates having been proposed⁴⁴ since the term EDHF was first coined in 1988⁴⁵. One of the putative EDHF candidates, at least in certain vascular beds, is C-type natriuretic peptide (CNP)⁴⁶. Our previous work has demonstrated that exogenous CNP prevents both leukocyte recruitment and platelet aggregation⁴⁷. Thus, it is tempting to speculate that CNP, as an EDHF, may underpin the anti-leukocyte, anti-atherogenic phenotype of the endothelium of females. Further investigation of the role of CNP in leukocyte recruitment and any sex differences that may be apparent is clearly warranted.

Early leukocyte rolling in vivo is mediated by the interaction of endothelial (particularly P-selectin) and leukocyte adhesion molecules (particularly PSGL-1). Our evidence suggests that whilst alterations in leukocyte adhesion molecules are unlikely to mediate the comparatively lower cell recruitment in females, changes in endothelial adhesion molecule expression might explain this phenomenon. Using primary endothelial cells isolated and cultured from male and female WT mice we demonstrated that whilst P-selectin expression is increased significantly in response to IL-1β treatment of endothelial cells of male mice, expression of this adhesion molecule was largely unchanged following treatment of endothelial cells from female mice. These results support the view that female sex hormones exert protection, at least in part, by modulating expression of P-selectin on endothelial cells during leukocyte rolling. Previous work from our laboratory has demonstrated that the reduction of leukocyte rolling exerted by CNP in mesenteric postcapillary venules is due to a suppression of P-selectin expression; an observation furthering a role for EDHF in the cytoprotective effects of female sex on leukocyte recruitment. A limitation of our findings, however, is that lung endothelial cells rather than cells of mesenteric origin were used to assess the P-selectin response to the cytokine. This was due to the difficulty in obtaining sufficient purity and numbers of cells from the venular side of the mesenteric circulation.

In summary, the present study demonstrates that leukocyte rolling is modulated by female sex hormones in the present of an inflammatory stimulus and that this effect is independent of both NO and PGI₂, and possibly due to EDHF activity. In turn, we propose that our evidence supports modulation of the expression of P-selectin on endothelial cells by sex hormones to prevent leukocyte rolling, which has downstream consequences for the extent of cell adhesion and emigration. A growing body of evidence suggests that leukocyte recruitment is likely to be pathogenic in atherosclerotic disease. Indeed, circulating neutrophil numbers and levels of neutrophil chemokines⁴⁸ are correlated with severity in acute coronary syndromes as well as being evident in thrombi and at sites of plaque and rupture in patients^49-51. In addition leukocytes have been identified at lesional sites in the early stages of plaque formation and neutrophil depletion is associated with decreased atherosclerotic load in mouse models of disease^52-55. The role of P-selectin in these phenomena is uncertain, especially since genetic deletion of this adhesion molecule appeared to have no impact on the beneficial effects of oestradiol treatment on atherosclerosis in a mouse model of disease⁵⁶, whilst VCAM-1 has been implicated^16;56. Although, the possibility that other adhesion molecules might have been upregulated and, therefore, compensated for the absence of P-selectin was not investigated. We suggest that since leukocyte recruitment has been implicated in CVD and our findings suggest that leukocyte recruitment is specifically attenuated in inflammation in females that these effects of sex on leukocyte recruitment may play a role in mediating the cardioprotection evident in pre-menopausal females.