Arbutin and decrease of potentially toxic substances generated in human blood neutrophils

Neutrophils, highly motile phagocytic cells, constitute the first line of host defense and simultaneously they are considered to be central cells of chronic inflammation. In combination with standard therapeutic procedures, natural substances are gaining interest as an option for enhancing the effectiveness of treatment of inflammatory diseases. We investigated the effect of arbutin and carvedilol and of their combination on 4β-phorbol-12β-myristate-13α-acetate- stimulated functions of human isolated neutrophils. Cells were preincubated with the drugs tested and subsequently stimulated. Superoxide (with or without blood platelets, in the rate close to physiological conditions [1:50]) and HOCl generation, elastase and myeloperoxidase release were determined spectrophotometrically and phospholipase D activation spectrofluorometrically. The combined effect of arbutin and carvedilol was found to be more effective than the effect of each compound alone. Our study provided evidence supporting the potential beneficial effect of arbutin alone or in combination with carvedilol in diminishing tissue damage by decreasing phospholipase D, myeloperoxidase and elastase activity and by attenuating the generation of superoxide and the subsequently derived reactive oxygen species. The presented data indicate the ability of arbutin to suppress the onset and progression of inflammation.


Introduction
Neutrophils are an important part of the innate host defense. Granules from viable human neutrophils contain a variety of enzymes known to be released into the extracellular milieu as a result of stimulation by natural (Wachtfogel et al., 1983) as well as artificial stimuli (Pečivová et al., 2004a). Elastase may be responsible for proteolysis of vital structures (Greene & McElvaney, 2009). Its release during blood coagulation independently of thrombin suggests participation of kallikrein in neutrophil activation and expands the appreciation of the role of elastase in human diseases (Wachtfogel et al., 1983). In the physiology of living systems, reactive oxygen species (ROS) and myeloperoxidase (MPO) are known to play a role which can be both beneficial (microbicidal;  (Petríková et al., 2002). It was found to inhibit luminolenhanced chemiluminescence of whole human blood in vitro (Nosáľ et al., 2005) and to decrease superoxide generation and MPO release from isolated human neutrophils activated with FMLP .
In combination with standard therapeutic procedures, natural substances are gaining interest as an option to enhance the effectiveness of treatment of inflammatory degenerative diseases. Plant-based extracts are usually well tolerated, have less severe side effects and synergistic or additive pharmacological effects (Rios et al., 2005;Matsuda et al., 1991). The compound of bearberry leaf (Arctostaphyllos uva-ursi), arbutin (ARB), a hydroquinone derivative, is able to protect plants against free radical mediated and enzymatic membrane lysis (Oliver et al., 1996). Besides its antiseptic and diuretic properties, ARB was reported to possess anti-inflammatory (Lee & Kim, 2012) and antioxidant effects (Takebayashi et al., 2010) and the ability to decrease leukocyte infiltration and MPO activity in the mouse skin model of tumour promotion (Nakamura et al., 2000). ARB was found to potentiate the antiinflammatory effects of indomethacin and corticosteroids (Rios et al., 2005;Matsuda et al., 1991), to decrease radical formation in human neutrophils in vitro and in experimental arthritis (Jančinová et al., 2007) and to decrease enzyme release from human neutrophils in vitro (Pečivová et al., 2011;Pečivová et al., 2012). ARB was reported to have antioxidant and other beneficial effects on the rat model of heart and mesenteric ischaemiareperfusion (Brosková et al., 2012).
In this study the impact of carvedilol, arbutin and their combination on ROS generation and degranulation was studied on the model of PMA-stimulated isolated neutrophils.

Isolation of blood platelets
Isolation of blood platelets was performed according to Nosáľ et al. (2000). Briefly, fresh blood was obtained at the blood bank by venepuncture from healthy male donors (20-50 years) without any medication for at least 7 days. Blood was anticoagulated with 3.8% trisodium citrate in the ratio 9:1 and centrifuged at 260 × g for 15 min. Platelet rich plasma (PRP) was removed, mixed with a solution containing 4.5% citric acid and 6.6% glucose (50 μl/ml PRP), and centrifuged at 1 070 × g for 10 min. Platelets were resuspended in an equal volume of Tyrode's solution (136.9 mmol/l NaCl, 2.7 mmol/l KCl, 11.9 mmol/l NaHCO 3 , 0.4 mmol/l NaH 2 PO 4 .2H 2 O, 1 mmol/l MgCl 2 .6H 2 O, and 5.6 mmol/l glucose) containing 5.4 mmol/l EDTA, pH 6.5. After 10-min stabilisation, the suspension was centrifuged for 6 min at 1 070 × g and platelets were resuspended in the same buffer without EDTA (pH 7.4) to obtain 5 × 10 5 platelets/μl.
Contaminating red blood cells were removed by hypotonic lysis. After centrifugation at 500 × g, 10 min, neutrophils were washed in PBS and resuspended in calcium-and magnesium-free PBS buffer in the final concentration of 1 × 10 7 /ml cells.

Superoxide determination
Superoxide formation was measured in isolated human neutrophils or in the mixture of neutrophils and blood platelets as superoxide dismutase inhibitable reduction of cytochrome c. Suspension (1 × 10 6 neutrophils or mixture neutrophils : blood platelets [1:50] / PBS with 0.9 mmol/l CaCl 2 , 0.5 mmol/l MgCl 2 ) was preincubated for 5 min at 37 o C with CAR and/or ARB [0.1-100 μmol/l] and subsequently stimulated by PMA [1 μmol/l] for 15 min at 37 °C. Controls were included for the effect of the stimulus, of CAR and ARB on cytochrome c reduction. After centrifugation 4 200 × g for 4 min at 4 °C, absorbance was measured at 550 nm in a microplate spectrophotometer (Labsystem Multiscan RC, MTX Labsystems, Inc., Vienna, Wyoming, USA).

Myeloperoxidase release
For determination of MPO release, neutrophils were preincubated with cytochalasin B [5 μg/ml] for 5 min at room temperature. Then the neutrophils (2x10 6 /sample) were preincubated with CAR and/or ARB [0.1-100 μmol/l] in a shaker bath at 37 °C for 5 min, followed by 15-min exposure to PMA. The activity of MPO was assayed in the supernatant after centrifugation 983 × g for 10 min at 4 °C by determining the oxidation of o-dianisidine in the presence of hydrogen peroxide in a microplate

Phospholipase D activity
We measured phospholipase D (PLD) activity by using the Amplex® Red PLD Assay Kit. It provides a sensitive method for measuring PLD activity in vitro. In this enzymecoupled assay, PLD activity is monitored indirectly using 10-acetyl-3,7-dihydrophenoxazine (Amplex Red reagent), a sensitive fluorogenic probe for H 2 O 2 . Fluorescence was measured on a microplate spectrofluorometer (TECAN infinite 200, Tecan Group Ltd. Männedorf, Switzerland) by excitation at 530 nm and emission detection at 590 nm.

Statistical analysis
Statistical significance of differences between means was established by Student's t-test and p-values below 0.05 were considered statistically significant.

Results
Isolated human neutrophils were stimulated by a soluble stimulus, PMA, which bypasses membrane receptors and activates NADPH-oxidase via protein kinase C (PKC).
Neither ARB nor CAR [0.1-100 μmol/l] had a significant effect on superoxide generation and enzyme release from unstimulated human isolated neutrophils. To compare the effects of the drugs tested, we expressed the results as p.c. of stimulus (control = 100%).
CAR or ARB alone dose dependently [0.1-100 μmol/l] decreased superoxide generation in neutrophils (results not shown). Figure 1 shows the lowered effect of each drug tested alone and the combined, more pronounced decreasing effect of ARB and CAR on PMA-stimulated superoxide generation in the mixture of neutrophils:platelets (1:50).
The respective effects of CAR alone, ARB alone and their cumulative effect on MPO, elastase and PLD are seen in Figure 2

Discussion
Neutrophils, highly motile phagocytic cells, constitute the first line of host defense, and simultaneously are considered to be central cells of chronic inflammation.
Acute inflammation is a protective highly coordinated sequence of events involving a large number of molecular, cellular and physiological changes. In a typical acute inflammatory response, effective clearance of microbial infections and/or damaged tissue is followed by resolution. This involves the elimination of neutrophils via programmed cell death, their phagocytosis by monocytederived macrophages that have been recruited to the  inflamed site following neutrophil influx, and in turn the clearance of these macrophages. If defects arise during any part of this highly conserved pathway, inflammation will persist and become chronic (Stables & Gilroy, 2011).
Chronic inflammatory diseases are characterised by overrecruitment of leukocytes, their interactions with endothelial and other inflammatory cells resulting in increased ROS generation, enzyme release and secretion of inflammatory cytokines and chemokines (Cascão et al., 2010;Serhan et al., 2008;Von Hundelshausen and Weber, 2007). Persistent and excessive activation of neutrophils, along with delayed apoptosis, can intensify the risk of tissue damage and have the potential to maintain permanent inflammation. Downregulation of activated neutrophil functions may thus significantly potentiate the effectiveness of standard therapy of diseases associated with chronic inflammation and reduce undesired effects.
In combination with standard therapeutic procedures, natural substances are gaining interest as an option to enhance the effectiveness of treatment of inflammatory diseases.
In this study, we measured superoxide generation, elastase and MPO release, HOCl generation, and PLD activity, factors able to participate during pathophysiological conditions in the disturbance of successful apoptosis and resolution of inflammation.
To mimic inflammatory conditions, we tested the potential protective effect of ARB and CAR alone and in combination on the model of PMA-activated human neutrophils. In our experimental conditions, CAR dosedependently decreased superoxide generation, similarly as recorded for fMLP-, PMA-and OZ-stimulated neutrophils (Asbrink et al., 2000;Yue et al., 1992;Mačičková et al., 2007). In human neutrophils, CAR interfered in vitro and ex vivo both with ROS generation and with already generated ROS, suggestive of its preventive and therapeutic effect (Drábiková et al., 2006), yet it was shown to be a poor scavenger of superoxide, as proven in a cell-free system (Asbrink et al., 2000;Nosáľ et al., 2005).
ARB dose-dependently decreased superoxide generation of isolated neutrophils in our experiments (results not shown), as well as chemiluminescence in whole human blood and external oxidant generation without affecting oxidative burst arising inside isolated human neutrophils (Jančinová et al., 2007).
Platelets play an important pro-inflammatory role, associated with adhesion of platelets to leukocytes and the vessel wall. The interaction of platelets with neutrophils promotes the recruitment of neutrophils into inflammatory tissue (Von Hundelshausen & Weber, 2007).
The effect of ARB and CAR on the decrease of PMAstimulated superoxide generation was lower in the mixture of neutrophils and blood platelets than in isolated neutrophils alone, and significant only in the higher concentration [100 μmol/l] used, yet the combination of the two drugs tested resulted in inhibition also in the lower concentration of CAR [10 μmol/l] (Figure 1). We ascribed the lower inhibitory effect of ARB and CAR on superoxide generation compared to their effects on chemiluminescence (Nosáľ et al., 2005) to their ability to "scavenge" free radicals derived from superoxide, since this ability of the drugs tested was reported also for other experimental models (Takebayashi et al., 2010;Lee & Kim, 2012) and was indicated by inhibition of MPO-derived HOCl in our experiments.
In vivo, blood platelets, accumulated and activated simultaneously with neutrophils, liberate substances that can eliminate or decrease the generation of oxygen metabolites, i.e. serotonin, β-thromboglobulin, adenosine and/or AMP. Mutual platelet-neutrophil interactions can weaken the effect of drugs (Ward et al., 1988;Pečivová et al., 2004b;Číž et al., 2007).  The inhibitory effect of CAR on superoxide generation and enzyme release from PMA-stimulated neutrophils in higher concentrations (10 and 100 μmol/l), in contrast to lower concentrations (Pekarová et al., 2009), indicates the possibility that CAR, similarly to other lipophilic β-adrenoceptor/blocking drugs, interferes with membrane structure, influencing predominantly phospholipid metabolism. Physicochemical properties of CAR (partition coefficient, dipole moment, and high lipophilicity) and its antiplatelet activity (Petríková et al., 2002) support this conclusion.
Phospholipases C and D have been linked to the antimicrobial functions of reactive oxidant generation, granule secretion, and phagocytosis (Gomez-Cambronero & Keire, 1998). Exposure of many cell types to PMA stimulates PLD activity, thereby implying an action by PKC. PLD hydrolyses phosphatidylcholine to phosphatidic acid (PA) and choline, where PA is considered to be the main effector of PLD function in cells. PA is reported to function as a second messenger, involved in membrane protein recruitment and membrane fusion processes, and PLD is proposed to play a role in signalling, intracellular transport, cytoskeletal rearrangements and apoptosis (Nozawa, 2002).
Since in our experiments ARB inhibited PMAstimulated but not the spontaneous superoxide generation and chemiluminescence (Jančinová et al., 2007), it seems to interact with some processes involved in activation of neutrophil oxidative burst.
We studied the effect of ARB, CAR and their combination on PLD activity, MPO and elastase release from granules of PMA-stimulated neutrophils. A higher inhibitory effect of CAR on MPO release from intact cells was recorded in comparison to its direct effect on enzyme activity . Both drugs decreased MPO and elastase release, and applied together, their effect was more pronounced (Figure 2 and 3). The same "trend" was measured for PLD activity (Fig.4). Based on the structural similarity of ARB to a known inhibitor of phospholipase A 2 , it appeard conceivable that inhibition of phospholipases (Oliver et al., 1996) or interference with phospholipid derived mediators (Stables & Gilroy, 2011) may be possible mechanisms involved.
Neutrophil apoptosis is one of the critical determinants of the outcome of the inflammatory response (Filep & El Kebir, 2009). Rapid induction of apoptosis in activated neutrophils and their subsequent removal from inflammatory sites by macrophages may prevent further damage to healthy tissues caused by ROS generation and release of toxic granule contents.
MPO triggers elastase and MPO from the granules and upregulates surface expression of Mac-1 on neutrophils, implying an autocrine and paracrine mechanism for perpetuation of the inflammatory response (Filep & El Kebir, 2009;Lau & Baldus, 2005). At sites of inflammation, MPO may function as a catalytic sink for NO, influencing its bioavailability (Abu-Soud & Hazen, 2000), and moreover, it is implicated to prolong the life span of neutrophils, thereby delaying the resolution of inflammation (Filep & El Kebir, 2009;Lau & Baldus, 2005).
Apoptotic neutrophils display changed morphological and biochemical characteristics and show molecular alterations on their cell surface. Resolution of inflammation is normally associated with orderly removal of dying apoptotic inflammatory cells by phagocytes through cell recognition receptors. Phosphatidylserine is a molecular marker whose externalisation facilitates the recognition of apoptotic neutrophils by macrophages (Akgul et al., 2001).
Flow cytometry demonstrated that neutrophil elastase cleaved the phosphatidylserine receptor, implying a potential mechanism for delayed apoptotic cell clearance in vivo (Vandivier et al., 2002).
The neutrophil serine protease elastase occupies an important position at the apex of a specific protease hierarchy. It has a number of important intrinsic proteolytic properties, it behaves also as a proinflammatory mediator, and in certain circumstances controls signalling mechanisms regulating innate immunity (Greene & McElvaney, 2009).
ARB was reported to have the potency to weaken priming of neutrophils during the inflammatory process, to decrease the concentration of radicals generated extracellularly by neutrophils, and that without affecting the formation of intracellular radicals essential for regulatory and microbicidal functions (Jančinová et al., 2007). Moreover, ARB has been suggested to regulate adhesion and trafficking of leukocytes and to attenuate LPS induced expression of inflammatory genes, such as iNOS, IL-1β, and TNF-α by downregulation of NF-κB (Lee & Kim, 2012).
The pluripotency of MPO and elastase distinguishes them as unique factors controlling many aspects of infection and inflammation. They are implicated as participants involved in direct cytotoxicity as well as in the perpetuation of inflammation. Thus the ability of ARB to decrease their free availability, along with reducing ROS generation, may significantly potentiate the effectiveness of therapy of diseases associated with chronic inflammation, reducing at the same time undesired effects of the antiinflammatory drugs used.
The presented data, together with those reported in the literature, indicate the beneficial ability of ARB to suppress the onset and progression of inflammation.