Chapter 141

Effects of Hydroxytyrosol on Macrophage Activation

Daniela De Stefano, Maria Chiara Maiuri and Rosa CarnuccioDipartimento di Farmacologia Sperimentale, Università degli Studi di Napoli ‘Federico II’, Napoli, Italy

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12Olives and Olive Oil in Health and Disease Prevention.ISBN: 978-0-12-374420-3

141.1  IntroductIon

Evidence from several human clinical studies suggests that polyphenolic compounds from extra virgin olive oil, present in the ‘Mediterranean diet’, exert protective effects on degenerative disorders in humans (Fitó et al., 2007). 3,4-Dihydroxyphenylethanol (hydroxytyrosol, HT), one of the major components in olive oil phenolic frac-tion, possesses a good bioavailability and its metabolism has been elucidated in animals and humans (Tuck and Hayball, 2002; Mirò-Casas et al., 2003). HT is a strong metal-chelator and scavenges a variety of endogenous and exogenous free radicals and oxidants (Visioli et al., 2002). In recent years, the interest in research on HT biological activities has grown. HT seems to contribute to the preven-tion of atherosclerosis and cancer pathogenesis (Visioli et al., 2002). The functional relationship between these pathological events and chronic inflammation is now widely accepted (Yan and Hansson, 2007; Zhang and Mosser, 2008). However, many of the molecular and cellular mech-anisms underlying this relationship remain poorly known. Activated macrophages, the bridge between innate and adaptive immunity, are considered major contributors to the initiation, propagation and resolution of inflammation. A failure of mechanisms required for resolving the inflamma-tory response can lead to chronic inflammation. A sustained accumulation of cytokines, growth factors, arachidonate metabolites, matrix metalloproteinases and free radicals produced by activated macrophages may cause alterations in critical pathways responsible either for vascular or DNA repair, contributing to the onset and progression of pathol-ogies such as atherosclerosis and cancer (Fujiwara and Kobayashi, 2005; Yan and Hansson, 2007). Many studies report the anti-inflammatory properties of whole polyphe-nolic compounds from extra virgin olive oil (Visioli et al., 2002; Bitler et al., 2005). A virgin olive oil with a high content of polyphenolic compounds exhibits protective

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effects on carrageenin-induced edema and adjuvant arthri-tis in rats (Martinez-Dominguez et al., 2001). Olive oil phenolic fraction reduces arachidonic acid mobilization and subsequent metabolism through the cyclooxygenase-2 (COX-2) pathway in phorbol 12-myristate 13-acetate (PMA)-stimulated rat peritoneal macrophages (Moreno et al., 2001). The administration of phenol-rich oils is dose- dependently associated with a decreased urinary excretion of 8-iso-prostaglandin F2α (PGF2α) in human volun-teers (Visioli et al., 2000a). Relatively few studies have addressed the molecular mechanisms responsible for HT anti-inflammatory effects, even less the macrophage activa-tion. HT inhibits platelet aggregation as well as proinflam-matory eicosanoid generation by 12-lipoxygenase in human serum (Petroni et al., 1995). Moreover, HT inhibits leuko-triene B4 formation by reducing the activity of the catalyz-ing enzyme 5-lipoxygenase (de la Puerta et al., 1999). Also, a low dose of HT is able to inhibit passive smoking induced oxidative stress in rats, as demonstrated by a reduced uri-nary excretion of PGF2α (Visioli et al., 2000b). A grow-ing body of evidence reports that inflammation plays a central role in the development of atherosclerotic disease, from the early phases of lesion formation to plaque rupture (Yan and Hansson, 2007). The major cause of persistent macrophage recruitment and activation within developing atherosclerotic plaques is oxidized forms of low-density lipoprotein (oxLDL) taken up by scavenger receptors (SRs) (Glass and Witztum, 2001). The protective effects of HT and other phenolic compounds from extra virgin olive oil against atherosclerosis have been extensively demon-strated. However, the molecular mechanisms by which these compounds exhibit their effects are not well known. The chronic intake of a ‘Mediterranean diet’ enriched in virgin olive oil decreases plasma concentrations of soluble vascular cell adhesion molecule-1 (VCAM-1) correlated with the inhibition of transcription factor nuclear factor-B (NF-B) activation in peripheral blood mononuclear cells

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from healthy volunteers (Perez-Martinez et al., 2007). Ex vivo observations showed that, in contrast to butter- and walnut-rich meals, the consumption of an olive-oil-rich food does not induce the postprandial activation of NF-B pathway in monocytes from healthy volunteers (Bellido et al., 2004). Moreover, a polyphenolic-enriched olive oil diet inhibits NF-B activation, but does not affect peroxi-some proliferator activated receptor-gamma expression, in PMA-stimulated monocyte/macrophages isolated from healthy volunteers (Brunelleschi et al., 2007). Peritoneal macrophages from mice, following an olive-oil-enriched diet, showed a lower level of mRNA for macrophage SRA type I, SRA type II and CD36 (Miles et al., 2001). In healthy volunteers immediately after the meal, phenolic compounds from extra virgin olive oil (tyrosol and HT) are present in all classes of plasma lipoproteins increas-ing their antioxidant capacity (Bonanome et al., 2000). HT prevents LDL oxidation in vitro (Grignaffini et al., 1994) and increases plasma antioxidant capacity in vivo (Jemai et al., 2008). The effectiveness of HT in counteracting the oxidative modifications of LDL has been reported in mouse macrophages (Di Benedetto et al., 2007). It is well known that oxLDL induce endothelial cells to produce potent monocyte activators as well as to increase adhesion molecule expression (Badimon et al., 2006). HT inhibits gene expression of adhesion molecules by blocking activa-tor protein-1 (AP-1) and NF-B as well as NADPH oxi-dase activation in endothelial cells (Carluccio et al., 2003, 2007; Dell’Agli et al., 2006). Also the connection between chronic inflammation and cancer induction has been inves-tigated (Karin et al., 2006). Toxic radicals and inflamma-tory mediators produced by activated macrophages have been implicated in the induction and development of this process (Federico et al., 2007). HT prevents chemically induced DNA and amino acid modifications in several human cell lines (Manna et al., 1997; Deiana et al., 1999; Schaffer et al., 2007). HT counteracts the cytotoxic effect of reactive oxygen metabolites on human blood mono-cytes preventing DNA and cell damage (Young et al., 2008). Here, we first describe some functions of macro-phages whose inappropriate activation might play a critical role in atherogenesis and carcinogenesis. Then, we show that HT is able to inhibit the activation of transcription fac-tors and related-gene expression, preventing macrophage activation.

141.2  Macrophage actIvatIon

Inflammation is a complex and well-coordinated response of an innate and adaptive immune system following infec-tion or injury. This process is characterized by a vascular response and recruitment of circulating leukocytes, defined initially by polymorphonuclear granulocytes followed by monocytes, which differentiate locally into macrophages

(Karin et al., 2006). Macrophage activation is induced through toll-like receptor (TLR) interaction by pathogen-associated molecular patterns, like lipopolysaccharide (LPS), and/or inflammatory mediators, like interferon- or oxLDL. This interaction initiates a biochemical signaling cascade leading to an increased expression of genes encod-ing for mediators, such as cytokines and chemokines, lipid metabolites, enzymes, adhesion molecules, reactive oxy-gen and nitrogen species (ROS/RNS) (Karin et al., 2006; Yan and Hansson, 2007). All of these mediators, in turn, amplify recruitment and activation of additional inflam-matory cells and are responsible for increased functions of activated macrophages (Figure 141.1). The predominant signaling pathways responsible for increased gene expres-sion result in the activation of NF-B, interferon regula-tory factors (IRFs), AP-1, cAMP-response element binding and signal transducer and activator of transcription (STATs) (Beutler, 2004). These transcription factors, binding to con-sensus sequences in the promoter region, can cooperate with one another to promote synergistically transcriptional activ-ity, up-regulating the activity of target genes (Ohmori et al., 1997). Tumor necrosis factor-α and interleukin-1 (IL-1), considered ‘early response cytokines’, up-regulate expression of adhesion molecules on endothelial cells thus facilitating phagocyte migration to sites of tissue injury. Other cytokines produced by macrophages, including IL-6, IL-12 and IL-18 promote type 1 helper T and type 2 helper T inflammatory/immune response (Fujiwara and Kobayashi, 2005). ROS and RNS play a crucial role against pathogens and tumor cells. ROS are produced by a number of cellular oxidative metabolic processes including oxidative phosphorylation by the mitochondrial respiratory chain, xanthine oxidase and NADPH oxidases. RNS include nitric oxide (NO) and per-oxynitrite. NO, a small free radical, generated by inducible nitric oxide synthase (iNOS) at high levels, is also an inducer or suppressor of apoptosis or immunoregulator. Nitrite and nitrate, stable metabolites of NO, can react rapidly with superoxide anion, forming peroxynitrite. ROS and RNS cause oxidative and nitrosative damage to nucleic acids, pro-teins, carbohydrates and lipids leading to a decrease of cel-lular proliferation and nucleic acid biosynthesis. Although there are intracellular defenses against ROS and RNS, both of them increase production or loss of antioxidant system lead-ing to progressive cell damage and decline in physiological function. COX-2 enzyme catalyzes the conversion of arachi-donic acid into PGs which, key mediators of pain and fever, induce the expression of inflammatory cytokines enhancing, in turn, ROS and RNS production. Overproduction of ROS and RNS as well as all of macrophage-derived mediators can also lead to tissue injury and toxicity in the host (Federico et al., 2007). Moreover, macrophages are not only involved in the initiation and propagation, but also in the resolution of inflammation. This is a finely coordinated and controlled process, in which locally inflammatory cells are eliminated via phagocytosis by macrophages. Activated macrophages

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chapter  |  141 Effects of Hydroxytyrosol on Macrophage Activation

FIgure 141.1  Onset and progression of inflammation.

exhibit several receptors on their surface, including vitronec-tin, phosphatidylserine, SRs, for the recognition and phago-cytosis of other cells, apoptotic cells or antigens. Among SRs, involved in the clearance of apoptotic cells, CD36 recognizes oxide sites on apoptotic cells that mimic oxLDL (Savill et al., 2002). There is growing evidence that clearance of apoptotic cells by phagocytosis can result in powerful anti-inflamma-tory and immunosuppressive effects, protecting tissue from harmful exposure to the inflammatory and immunogenic contents of dying cells. The resolution of inflammation is due also to production of anti-inflammatory mediators, such as IL-10 and transforming growth factor- (TGF-), in the lesion by tissue macrophages that phagocytosed apoptotic cells. IL-10 potently inhibits the expression of cytokines and soluble mediators. TGF- reduces cellular proliferation and differentiation. Furthermore, increased levels of TGF- are correlated with an augmentation of phagocytosis (Figure 141.2). Disorder of apoptosis leading to leukocyte survival, defective clearance of apoptotic cells as well as inappropri-ate macrophage activation have been suggested to contribute, at least in part, to the development of chronic inflammation (Savill et al., 2002). Finally, due to the nature of their potent signaling of the innate immune response, macrophages can influence almost every aspect of the inflammatory process.

141.2.1  Macrophage activation and atherogenesis

Atherosclerosis is a chronic metabolic disease characterized by lipid accumulation and inflammation (Yan and Hansson,

2007). The role of activated macrophages in the formation of the atherosclerotic plaque has been extensively reviewed (Glass and Witztum, 2001). The earliest process of athero-sclerosis is the retention of deposited LDL in the intima of arteries and, subsequently oxidized, they form oxLDL. In response to oxLDL stimulation, endothelial cells express adhesion molecules, including VCAM-1 and intercellular adhesion molecule-1, and secrete chemokines, leading to the recruitment of monocytes into the intima. Monocytes

FIgure 141.2  Resolution of inflammation.

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differentiate into macrophages which express SRs and TLRs. SRs mediate macrophage uptake of oxLDL particles, leading to the intracellular cholesterol accumulation and for-mation of foam cells. TLRs, predominantly TLR2 or TLR4, in collaboration with co-receptors and SRs, interacting with oxLDL or microbial components, such as LPS and other ligands, induce various signaling pathways increasing gene expression in macrophages. TLR2 or TLR4 ligands elicit, mainly, two signaling cascades leading to activation of NF-B or IRF-3, which regulate the expression of several genes encoding for bioactive molecules, such as cytokines, chemo-kines, growth factors, matrix metalloproteinases (MMPs), adhesion molecules, eicosanoids and ROS/RNS (Yan and Hansson, 2007). These mediators, produced by activated macrophages, influence the function of vascular cells. High concentrations of iNOS-derived NO can have cytotoxic effects on arterial cells, cause apoptosis and further induce the expression of proinflammatory genes. It is also impor-tant to note that, in addition to increased iNOS expression during lesion development, superoxide anion production is stimulated by hypercholesterolemia in endothelial cells, and can lead to increased peroxynitrite formation. RNS can rap-idly oxidize lipoproteins and cause several harmful effects in the arterial wall (Luoma and Ylä-Herttuala, 1999). A marked increase in expression of COX-2 and MMP (MMP-2 and MMP-9) enzymes is likely an important pathogenic mechanism responsible for plaque destabilization (Cipollone et al., 2005). In addition, macrophages and dendritic cells also activate T cells via antigen presentation and modulate T-cell responses by production of cytokines, such as IL-18 and IL-12. Altogether these inflammatory mediators, in con-cert with T cell produced cytokines, promote inflammatory responses, stimulate the lesion progression and increase the risk of plaque rupture (Yan and Hansson, 2007).

141.2.2  Macrophage activation and carcinogenesis

The chronic infiltration of activated macrophages into tis-sue contributes to the onset of cancer, its development and progression (Zhang and Mosser, 2008). A sustained generation of cytokines, ROS and RNS, produced by acti-vated macrophages, can cause a variety of DNA altera-tions including oxidative and nitrosative damage on critical genes and proteins involved in maintaining normal cel-lular homeostasis, leading to a protumorigenic micro-environment (Lala and Chakraborty, 2001). Macrophage infiltration is involved in tumor development, such as enhanced cell survival, tissue remodeling, angiogenesis and suppression of antitumor adaptive immune responses. An accumulation of macrophages has been associated with the production of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth fac-tor, which promote endothelial cell migration and tumor

neovascularization (Lala and Chakraborty, 2001). Moreover, macrophages produce tumor-inducing chemokines, including monocyte chemoattractant protein-1, and enzymes, including MMPs and COX-2. MMPs regulate cell behaviour and cell–cell communication. Enhanced COX-2 expression has been found in human invasive carcinomas and correlated with high levels of MMPs and VEGF (Lala and Chakraborty, 2001; Coussens and Werb, 2002). All of these mediators are regulated at the transcriptional level, mainly by NF-B, a potential therapeutical target because of its sustained activa-tion in many types of cancers (Karin and Greten, 2005). As cancers progress, however, tissue infiltrating macrophages also produce anti-inflammatory cytokines, such as IL-10 and TGF-, thus inducing a state of immunosuppression that prevents tumor rejection (Karin et al., 2006). These evidences strongly support the idea that activated macro-phages orchestrate the inflammatory network expressed in the tumor microenvironment.

141.3  ht InhIbItS Macrophage actIvatIon

The effects of HT on macrophage activation are illustrated in detail by results obtained in our laboratories. HT down-regulates iNOS and COX-2 gene expression by preventing NF-B, STAT-1α and IRF-1 activation mediated through LPS-induced ROS generation in J774 macrophages (Maiuri et al., 2005). We have shown that HT caused a significant and concentration-dependent inhibition of nitrite and PGE2 production in J774 macrophages stimulated with LPS for 24 h (Table 141.1). This effect exhibited by HT was not due to direct inhibition of either iNOS and COX-2 enzymatic activity. In fact, HT failed to reduce nitrite and PGE2 gen-eration when added to cells 12 h after LPS stimulation, suggesting that HT is not able to affect iNOS and COX-2 activity once the enzymes have been expressed. It is wor-thy of note to observe that inhibition of LPS-mediated nitrite and PGE2 production by HT correlated with a marked reduction either of iNOS or COX-2 protein expression and mRNA levels (Figure 141.3). LPS is a potent macrophage-activating stimulus that, interacting with TLR4, initiates an ordered recruitment of adaptor molecules which, mainly via Mal, MyD88 as well as Janus Kinases, lead to NF-B, STATs and IRFs activation (Beutler, 2004). These transcrip-tion factors can cooperate with one another to promote syn-ergistically transcriptional activity binding to consensus sequences in the promoter region and up-regulating iNOS and COX-2 gene expression (Blanco et al., 2000; Janabi, 2002; Teng et al., 2002). Interestingly, HT was able to affect NF-B, STAT-1α and IRF-1 activation in J774 macrophages stimulated with LPS (Figure 141.4), suggesting that HT inhibits iNOS and COX-2 gene expression by preventing transcription factor activation. These transcription factors are sensible to the intracellular redox state (Lee et al., 2007).

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Table 141.1 Effects of HT on nitrite and PGE2 production in LPS-stimulated J774 macrophages for 24 h.

HT added to the cells 10 minutes before LPS challenge

Production of mediators

Unstimulated cells

LPS (1 g mL1)

LPS (1 g mL1) HT (50 M)

LPS (1 g mL1) HT (100 M)

LPS (1 g mL1) HT (200 M)

NO2

(nmol/106 cells)

0.80 0.11 66.70 0.80 65.47 1.60 50.70 1.80** 30.10 1.10***

PGE2 (ng mL1) 1.00 0.70 25.10 0.90 22.40 0.80 17.40 0.60** 7.90 0.50***

HT added to the cells 12 h after LPS challenge

NO2

(nmol/106 cells)

0.50 0.09 66.0 0.50 64.9 1.20 67.50 1.0 65.30 0.60

PGE2 (ng mL1) 1.10 0.90 25.0 0.70 24.90 1.0 23.80 0.80 24.50 0.60

NO generation in the incubation medium was measured as nitrite (NO2) by Griess reaction. PGE2 accumulation in the culture medium was

measured by enzyme linked immunosorbent assay. Data are extrapolated from Maiuri et al., 2005 and expressed as means SEM of 3 separate experiments in triplicate.**p 0.01***p 0.001 vs. LPS alone.

HT significantly inhibited the LPS-induced ROS formation (Table 141.2) that was correlated to the inhibition of NF-B, STAT-1α and IRF-1 activation in J774 macrophages. Although we did not investigate the molecular mechanism by which HT prevents the activation of three transcription factors in activated macrophages, this effect is likely to be correlated to its antioxidant properties. Indeed, HT exhib-ited inhibitory effects when it was added to cells before, but not after LPS challenge (Table 141.1). This finding

FIgure 141.3  Effects of HT on iNOS and COX-2 protein and mRNA levels. HT inhibits iNOS and COX-2 protein expression (A) and mRNA levels (B). Results are from a densitometric analysis of Western blot and polymerase chain reaction, respectively. J774 macrophages were stimu-lated with LPS (1 g mL1) for 24 h (A) and 6 h (B) in the presence of HT (200 M). Data are extrapolated from Maiuri et al., 2005 and expressed as (% inhibition) SEM of 3 separate experiments. ***p 0.001 vs. control (LPS alone).

suggests that antioxidant HT acts at an early step of the LPS-induced signaling cascade leading to NF-B, STAT-1α and IRF-1 activation. Our results show that HT, by affect-ing NF-B, STAT-1α and IRF-1 activation, down-regulates iNOS and COX-2 gene expression. An up-regulated expres-sion of both enzymes in activated macrophages has been positively associated with atherosclerosis and cancer

FIgure 141.4  Effects of HT on activation of STAT-1α, IRF-1 and NF-B transcription factors. HT inhibits STAT-1α, IRF-1 and NF-B DNA-binding activity. Results are from a densitometric analysis of elec-trophoretic mobility shift assay. J774 macrophages were stimulated with LPS (1 g mL1) for 24 h in the presence of HT (200 M). Data are extrap-olated from Maiuri et al., 2005 and expressed as (% inhibition) SEM of 3 separate experiments. ***p 0.001 vs. control (LPS alone).

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Table 141.2 Effects of HT on intracellular ROS production in J774 macrophages stimulated with LPS for 24 h.

Production of ROS

Unstimulated cells

LPS (1 g mL1) LPS (1 g mL1) HT (50 M)

LPS (1 g mL1) HT (100 M)

LPS (1 g mL1) HT (200 M)

Relative fluorescence units

12.90 1.10 26.40 1.30 25.12 1.80 18.48 1.45** 12.93 1.36***

HT was added to the cells 10 min before LPS challenge. The formation of ROS was evaluated by using the probe 2,7-dichlorofluorescin-diacetate. Data are extrapolated from Maiuri et al., 2005 and expressed as means SEM of 3 independent experiments (n 10).**p 0.01.***p 0.001 vs. LPS alone.

(Luoma and Ylä-Herttuala, 1999; Lala and Chakraborty, 2001; Cipollone et al., 2005). iNOS-derived NO underlies vascular inflammation in atherogenesis, contributing to the onset of fatty streak and lesion progression (Luoma and Ylä-Herttuala, 1999). COX-2-derived PGE2 have been reported to induce MMP expression, involved in plaque disruption (Cipollone et al., 2005). Moreover, a sustained induction of iNOS expression may be potentially carcinogenic via NO-mediated DNA damage. NO can also promote tumor pro-gression by COX-2 activation (Lala and Chakraborty, 2001), nevertheless, studies addressing the interactions between iNOS and COX-2 are controversial. An increased COX-2 expression has been correlated with high levels of MMPs and VEGF in several human invasive cancers (Coussens and Werb, 2002). Our results demonstrating that HT reduces iNOS and COX-2 gene expression, blocking NF-B, STAT-1α and IRF-1 transcription factor activation, suggest for this compound a key role in the prevention of atherogenesis and carcinogenesis. Few studies have investigated the role of HT on macrophage activation at the molecular level. We demon-strated that HT, beside NF-B, inhibits STAT-1α and IRF-1 activation in LPS-stimulated macrophages. Inhibition of NF-B activation by HT has also been reported in endothelial cells (Carluccio et al., 2003, 2007; Dell’Agli et al., 2006). It is now becoming clear that the long-term use of non-steroidal anti-flammatory drugs, such as salicylates, as well as natural compounds from the ‘Mediterranean diet’, such as lycopene, quercetin and resveratrol (all inhibitors of NF-B activa-tion) not only suppresses the inflammatory response, but also reduces the incidence of atherosclerosis and many types of cancer (Kopp and Ghosh, 1994; Kaliora et al., 2006; Von Löw et al., 2007). Our observations suggest that HT, sharing the ability to inhibit NF-B activation in activated macrophages, may be included among these compounds. Furthermore, in vitro and in vivo studies are necessary to understand the molec-ular mechanisms by which HT elicits the protective effects. In conclusion, the potency of HT as an antioxidant suggests that it could be fruitfully employed as a prophylactic and/or poten-tial non-toxic agent for the control of inflammation and, ulti-mately, play a pivotal role as a component of extra virgin olive oil in atherosclerosis and cancer preventative action.

acknowledgMentS

The authors thank Professor Raffaele Sacchi for his helpful advices and comments. Dr Daniela De Stefano was supported by the Italian Society of Pharmacology (SIF).

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