Magnolol inhibits Mac-1 (CD11b/CD18)-dependent neutrophil adhesion: Relationship with its antioxidant effect

Abstract

Magnolol, a phenolic compound isolated from a Chinese herbal drug, Magnolia officinalis, has been shown to protect rat heart from ischemia/reperfusion injury. Neutrophil adhesion plays a crucial process during this inflammatory response. To evaluate whether magnolol prevents ischemia/reperfusion injury by inhibiting neutrophil adhesion, we determined whether magnolol can inhibit adhesion of phorbol-12-myristate-13-acetate (PMA)-activated human neutrophils to a fibrinogen-coated surface in a dose-dependent manner. Using flow cytometric analysis, we observed that magnolol pretreatment (10 min at 37°C) diminished PMA (100 ng/ml)-induced Mac-1 upregulation. PMA also induced rapid intracellular accumulation of superoxide (O₂^−·) and hydrogen peroxide (H₂O₂) in neutrophils; magnolol pretreatment attenuated the accumulation of these two substances. Inhibition of reactive oxygen species by superoxide dismutase and/or catalase, which decompose O₂^−· and H₂O₂, respectively, also abolished Mac-1 upregulation and neutrophil adhesion. We conclude that magnolol inhibits neutrophil adhesion and that this can account for its anti-ischemia/reperfusion injury effect. We propose that the inhibitory effect of magnolol on neutrophil adhesion to the extracellular matrix is mediated, at least in part, by inhibition of the accumulation of reactive oxygen species, which in turn suppresses the upregulation of Mac-1 that is essential for neutrophil adhesion.

Introduction

Magnolol, an active principle isolated from the Chinese herb `Houp,u' (Magnolia officinalis), has been shown to be an anti-platelet aggregation (Teng et al., 1988), vessel dilation (Teng et al., 1990) and anti-inflammation (Wang et al., 1992) agent. The anti-inflammatory effects of magnolol can be accounted for by its multiple pharmacological activities including inhibition of prostaglandin D₂ formation (Wang et al., 1992), suppression of non-selective vascular hyporeactivity to mediators (Wang et al., 1993), reduction of the formation of eicosanoids mediators (Wang et al., 1995) as well as its antioxidant potential (Lo et al., 1994; Chang et al., 1994; Chan et al., 1996) and its ability to prevent ischemic-reperfusion injury (Hong et al., 1996). Despite the numerous studies that have been performed, the mechanisms involved in these anti-inflammatory effects, especially the ischemic-reperfusion injury prevention effect, of magnolol remain unclear.

Recruitment of neutrophils into areas of inflammation is a key event during inflammatory responses. This begins with the binding of neutrophils to the endothelium, followed by their transmigration into tissues (Albelda et al., 1994). Neutrophil binding to the endothelium is characterized by distinct phases, including rolling, activation and firm adhesion (Ley, 1996). The molecular basis for these phases is the upregulation of various cell adhesion molecules that belong to three major families: (1) the selectins (e.g. P-selectin, L-selectin and E-selectin) (Bevilacqua and Nelson, 1993; Lefer et al., 1994), (2) the β2 integrins (e.g. CD11/CD18) (Ruoslahti, 1991) and (3) the immunoglobulin superfamily (e.g. intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and platelet-endothelial cell adhesion molecule-1) (Springer, 1990). While the selectins are important for rolling, firm adhesion of neutrophils is essentially β2 integrin dependent (Springer and Anderson, 1986; Arfors et al., 1987). The β2 integrins comprise a group of heterodimeric glycoproteins, including CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1) and CD11c/CD18 (p150,95), with LFA-1 and Mac-1 being the major integrins on neutrophils. Although all three of these β2 integrins may participate in the intercellular adhesive interaction, Mac-1 appears to be the principal form accountable for the firm adhesion of neutrophils (Lefer and Lefer, 1996). Increased neutrophil adhesion has been demonstrated to be an important factor in the pathogenesis of vascular injury during inflammatory processes, such as ischemia/reperfusion injury of the heart (Lefer, 1995), liver (Monden et al., 1995) and brain (Matsuo et al., 1994). Hence, therapeutic interventions targeting these phases of neutrophil adhesion may prove to be effective. This can be illustrated by the observation that administration of a monoclonal antibody directed against CD11b (Mac-1) could prevent intestine ischemia/reperfusion-induced lung injury, regardless of the activation state of neutrophils (Koike et al., 1995). However, it has been demonstrated that both superoxide and hydrogen peroxide can modulate leukocyte Mac-1 expression and leukocyte endothelial adhesion, an effect which can be diminished by superoxide dismutase and/or catalase pretreatment (Fraticelli et al., 1996; Serrano et al., 1996). Given the anti-oxidant potential of magnolol (Chang et al., 1994; Lo et al., 1994; Chan et al., 1996), whether magnolol can inhibit neutrophil adhesion as a mechanism to prevent ischemia/reperfusion injury, therefore, warrants further investigation.

We hypothesize that inhibition of ischemia/reperfusion injury by magnolol may be mediated by interference with the upregulation of adhesion molecules, leading to inhibition of neutrophil adhesion. As mentioned above, firm adhesion of neutrophils to the endothelium and to a large array of extracellular matrix proteins is primarily Mac-1 dependent (Beatty et al., 1983; Lefer and Lefer, 1996). Adhesion of phorbol-12-myristate-13-acetate (PMA)-activated neutrophils to a fibrinogen-coated surface was measured to investigate the effects of magnolol. We also examined the expression of Mac-1 on the surface of neutrophils after magnolol treatment, since both the expression and activation of Mac-1, through conformational change, have been shown to be important in the firm adhesion of neutrophils (Albelda et al., 1994; Ley, 1996). Moreover, because it has been shown that reactive oxygen species (such as superoxide, O₂^−·, and hydrogen peroxide, H₂O₂) can stimulate the translocation of Mac-1 from intracellular stores to the cell surface (Simms and D'amico, 1995), we examined the role of reactive oxygen species inhibition in the anti-adhesive effect of magnolol.