17β-oestradiol reduces cardiac leukocyte accumulation in myocardial ischaemia reperfusion injury in rat

Abstract

We investigated whether oestrogens modulate the phenomenon of leukocyte accumulation during ischaemia and reperfusion of the myocardium. Anaesthetized rats were subjected to total occlusion (1 h) of the left main coronary artery followed by 1 h reperfusion. Sham myocardial ischaemia–reperfusion rats (Sham) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity, serum creatinine phosphokinase activity, serum and macrophages tumour necrosis factor (TNF-α) and the myocardial staining of intercellular adhesion molecule-1 (ICAM-1) were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum creatinine phosphokinase activity (348±38 U/ml) and cardiac myeloperoxidase activity, a marker of polymorphonuclear leukocyte accumulation, both in the area at risk and in the necrotic area (MPO; 9±1.1 U×10⁻³/g tissue and 8.2±1 U×10⁻³/g tissue, respectively), and induced a marked increase in the macrophage (156±14 U/ml at the end of reperfusion) and serum (344±12 U/ml, at the end of reperfusion) levels of TNF-α. Finally, myocardial ischaemia–reperfusion injury increased ICAM-1 staining in the myocardium. Administration of 17β-oestradiol (5, 10 and 20 μg/kg, i.m. 5 min after induction of myocardial ischaemia–reperfusion injury), lowered myocardial necrosis and myeloperoxidase activity in the area at risk and in the necrotic area, reduced serum and macrophages TNF-α (20±3 U/ml and 9±3 U/ml, respectively) and decreased serum creatinine phosphokinase activity (67±3 U/ml). Oestrogen treatment also blunted the increased staining of ICAM-1 in the injured myocardium. Finally, 17β-oestradiol added in vitro to peritoneal macrophages collected from untreated rats subjected to myocardial ischaemia–reperfusion injury, significantly reduced TNF-α production. Our results suggest that 17β-oestradiol, by inhibiting TNF-α production, limits the deleterious ICAM-1-mediated binding of leukocytes to injured mycardium and protects against myocardial ischaemia–reperfusion injury.

Introduction

Several lines of evidence suggest that oestrogens are cardioprotective (Barrett-Connor and Bush, 1991). Although the cardioprotective benefits of oestrogens appear well established, the mechanism of this effect remains unclear and is the subject of intense investigation (Grady et al., 1992).

Alterations in plasma concentrations of lipoproteins, hemostatic factors, glucose and insulin and reductions in arterial blood pressure (Wahl et al., 1983; Stampfer and Colditz, 1991; Mcade and Berra, 1992) have been proposed as possible explanations for the oestrogen-induced cardioprotection. However, these factors alone cannot explain the positive effects of oestrogens on the cardiovascular system.

The involvement of an inflammatory response in the pathophysiology of myocardial ischaemia has also been suggested (Lucchesi, 1990). Leukocyte accumulation in the myocardium may amplify tissue damage by producing cell activation of the myocytes and by releasing deleterious substances such as leukotrienes (Feuerstein, 1984), thromboxane A₂ (Coker and Parrat, 1985), oxygen free radicals (McCord, 1985) and platelet activating factor (Braquet et al., 1987).

Adhesion molecules are considered to play a pivotal role in the localization and development of an inflammatory reaction. Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule normally expressed at a low basal level on endothelial cells, but its expression can be enhanced by several inflammatory mediators such as Interleukin-1 and tumour necrosis factor (TNF-α) (Wertheimer et al., 1992).

Structurally, ICAM-1 is a member of the immunoglobulin (Ig) supergene family with five Ig-like domains, a single transmembrane region and a short cytoplasmatic tail (Simmons et al., 1988). It is a ligand for at least two members of the CD18 family of leukocyte adhesion molecules: LFA-1 (CD11a/CS18) and Mac-1 (CD11b/CD18) (Marlin and Springer, 1987; Diamond et al., 1990).

Previous findings have suggested that cardiac myocytes express ICAM-1 in response to cytokine stimulation and that ICAM-1 serves as an adhesive molecule for neutrophils on this cell type (Smith et al., 1991). Furthermore, passive immunization with specific antibodies raised against ICAM-1 reduces infarct size and myocardial leukocyte accumulation in an experimental model of myocardial ischaemia in the rat (Ioculano et al., 1994).

In light of these findings we studied whether the cardioprotective effects of oestrogens may be mediated in vivo by a reduction in ICAM-1-mediated leukocyte accumulation in the ischaemic myocardium and therefore we investigated the effects of 17β-oestradiol on the pathological sequelae associated with occlusion and reperfusion of the myocardium in the rat.