Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil ﬁrm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium inﬂux Yea-Hwey Wang a , Wen-Yen Wang b , Jyh-Fei Liao a , Chieh-Fu Chen a,d , Yu-Chang Hou c , Kuo-Tong Liou f , Yueh-Ching Chou g,h , Jung-Hsiung Tien g , Yuh-Chiang Shen d,e,* a Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan b Departments of Surgery, Tao-Yuan General Hospital, Department of Health, Taiwan c Chinese Medicine, Tao-Yuan General Hospital, Department of Health, Taiwan d National Research Institute of Chinese Medicine, Taipei, Taiwan e Institute of Biological Sciences, College of Life Science, National Chung-Hshing University, Taichung, Taiwan f Department of Chinese Martial Arts, Chinese Culture University, Taipei, Taiwan g Department of Pharmacy, Taipei Veterans General Hospital, Taipei, Taiwan h School of Pharmacy, Taipei Medical University, Taipei, Taiwan Received 30 September 2003; accepted 10 February 2004 Abstract Taxifolin has been reported to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1), a receptor-mediating ﬁrm adhesion with b2 integrin (e.g., Mac-1) expressed on leukocytes. To evaluate whether taxifolin could modulate Mac-1-dependent ﬁrm adhesion by neutrophils, and the possible mechanism(s) underlying its anti-inﬂammatory action, its effects on N-formyl-methionyl- leucyl-phenylalanine (fMLP) or phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were studied. Pretreat- ment with taxifolin (1–100 mM) concentration-dependently diminished fMLP- or (PMA)-induced Mac-1-dependent ﬁrm adhesion and upexpression of surface Mac-1. Mobilisation of intracellular calcium and production of reactive oxygen species (ROS) signal the upexpression of Mac-1 and ﬁrm adhesion by neutrophils. Taxifolin impeded the calcium inﬂux induced by fMLP (a receptor-mediated activator) or AlF 4  (a G protein-mediated activator). Taxifolin also effectively inhibited the fMLP- or PMA-induced ROS production with 50% inhibitory concentration (IC 50 ) less than 10 mM, possibly through impairing the activation of NADPH oxidase, a major ROS- generating enzyme in neutrophils, by restricting the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC). In conclusion, we propose that impairment of ROS production by NADPH oxidase through interfering with p38 MAPK- and/or PKC-dependent signals, and antagonism of G protein-mediated calcium inﬂux may account for the inhibition of Mac-1-dependent neutrophil ﬁrm adhesion that confers taxifolin the anti-inﬂammatory activity. # 2004 Elsevier Inc. All rights reserved. Keywords: Calcium; Mac-1 (CD11b/CD18); NADPH oxidase; p38 Mitogen-activated protein kinase; Protein kinase C; Taxifolin 1. Introduction Plant ﬂavonoids have long been reported to inhibit the functions of human inﬂammatory cells, possibly through modulation of enzyme systems related to inﬂammatory responses and/or scavenging of reactive oxygen radicals. Of these, quercetin is the most documented [1]. Taxifolin (Fig. 1), with structure similar to that of quercetin, has been Biochemical Pharmacology 67 (2004) 2251–2262 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2004.02.020 Abbreviations: fMLP, N-formyl-methionyl-leucyl-phenylalanine; Mac- 1, macrophage adhesion molecule-1; p38 MAPK, p38 mitogen-activated protein kinase; PMA, phorbol-12-myristate-13-acetate; PKC, protein kinase C; ROS, reactive oxygen species * Corresponding author. Tel.: þ886-2-28201999x9101; fax: þ886-28264266. E-mail address: yuhcs@nricm.edu.tw (Y.-C. Shen).