Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp

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Abstract

The tamarind (Tamarindus indica L.) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC50 (in μg/106cells) = 115.7 ± 9.7 (LumCL) and 174.5 ± 25.9 (LucCL)], than the OZ- [IC50 = 248.5 ± 23.1 (LumCL) and 324.1 ± 34.6 (LucCL)] or fMLP-stimulated cells [IC50 = 178.5 ± 12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O2 consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 μg/106 cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases.

Introduction

Neutrophils, which are the most abundant leukocytes in the circulation, constitute the first line of defense against microorganisms, virally infected cells and tumor cells (Selvatici et al., 2006). Neutrophil activation by soluble or particulate stimuli leads to generation of reactive oxygen species (ROS) through the oxidative metabolism, which involves activation of the NADPH oxidase enzymatic complex and increase in cellular oxygen consumption (Tauber et al., 1984, Babior, 2000). In addition, these cells release proteolytic enzymes, such as elastase and cathepsins, that act as microbicidal agents, degrade the extracellular matrix and contribute to cellular migration at the inflammatory site (Lee and Downey, 2001, Braga et al., 2006).

Despite its fundamental role in the host defense, increased neutrophil recruitment and activation has been implicated in the pathogenesis of autoimmune and inflammatory diseases, such as systemic lupus erythematosus, rheumatoid arthritis, emphysema and Chron’s disease (Babior, 2000, Marzocchi-Machado et al., 2002, Jancar and Crespo, 2005). In the last decade, it has become clear that atherosclerosis is also an inflammatory disease, which involves hyperlipidemia, low-density lipoprotein (LDL) oxidation, increased oxidative stress and leukocyte recruitment and infiltration within the atheroma (Ross, 1999, Libby, 2002). However, the neutrophil role in the atherosclerosis pathogenesis is not completely understood. Its participation has been suggested in the early stages of the disease, by generating ROS that oxidize LDL, as well as contributing to the amplification of the inflammatory process in the late stages of the disease’s progression by releasing pro-inflammatory cytokines (Ricevuti, 1997, Shoenfeld et al., 2001). A strong correlation among high serum LDL levels, neutrophil activation and leukocytosis in atherosclerosis patients was reported by Chan et al. (1998).

In recent years, research papers have suggested that some drugs used in the therapy of autoimmune and inflammatory disorders can be successful for the treatment and/or prevention of atherosclerosis (Sherer and Shoenfeld, 2002). The close and complex relationship between atherosclerosis, inflammation and oxidative stress has been guiding the search for therapeutic strategies with anti-inflammatory and antioxidant compounds to reach beneficial effects in the prevention of atherosclerosis progression (Parker et al., 1995, Kourounakis et al., 2002). Phenolic-rich foods, beverages and plant extracts, such as red wine, grape seed extract, green tea, Lychnophora spp. and Tamarindus indica, seem to be promising sources of compounds with such characteristics, since they have shown hypolipidemic, anti-atherosclerotic, antioxidant, anti-inflammatory and immunomodulatory effects (Riemersma et al., 2001, Kanashiro et al., 2004, Vinson et al., 2004, Auger et al., 2005, Gobbo-Neto et al., 2005, Martinello et al., 2006, Landi-Librandi et al., 2007, Kanashiro et al., 2009).

Tamarindus indica L., a tree-type plant belonging to the Leguminosae (Caesalpiniaceae) family, occurs in the tropical regions of the world, and can be found in more than 50 countries. The major areas of production are the Asian countries like India, Bangladesh, Sri Lanka, Thailand, and Indonesia, and the African and the American continents (Sudjaroen et al., 2005, Kumar and Bhattacharya, 2008). Its fruits (tamarind), rich in sugars, organic acids, pectins, vitamins and minerals, have been used as spices and food components (Carasek and Pawliszyn, 2006). In traditional folk medicine, mainly in Asian countries, the T. indica fruits and seeds have had antimicrobial, digestive, laxative, expectorant, antidiabetogenic and immunomodulator uses (Komutarin et al., 2004, Maiti et al., 2004, Muthu et al., 2005, Ushanandini et al., 2006, Al-Fatimi et al., 2007, Sudjaroen et al., 2005).

Recent studies from our research group reported that the T. indica fruit pulp extract (ExT), rich in polyphenolic compounds, reduced the atherosclerosis progression and the oxidative stress in an experimental model of atherosclerosis in hamsters, and also showed in vitro antioxidant effect (Martinello et al., 2006). The ExT was also able to modulate the activity of the complement system in vitro and in vivo, which is an important immunomodulatory effect of this extract underlying inhibition of atherosclerosis progression (Landi-Librandi et al., 2007).

Considering the relationship among neutrophils, inflammation and atherosclerosis, the aim of this study was to investigate the modulatory effect of ExT on some human neutrophil effector functions, such as the oxidative metabolism triggered by different signaling pathways, and the cellular oxygen consumption, degranulation and elastase activity. The cytotoxic potential of ExT on neutrophils was also evaluated.

Section snippets

Chemicals

Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, cat.# A8511), lucigenin (bis-N-methylacridinium nitrate, cat.# M8010), zymosan A (cat.# Z4250), phorbol-12-myristate-13-acetate (PMA, cat.# P8139), cytochalasin B (CB, cat.# C6762) and trypan blue (cat.# 72-57-1) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The peptidic elastase substrate N-succinyl-alanine-alanine-valine-p-nitroanilide (SAAVNA, cat.# 454454) and n-formyl-methionyl-leucyl-phenylalanine (fMLP, cat.# 05-22-2500)

Cellular chemiluminescence inhibition

The superoxide anion and total ROS production by the stimulated neutrophils were measured by the lucigenin (LucCL)- and luminol (LumCL)-enhanced chemiluminescence assays, respectively. The three stimuli used, OZ, PMA and fMLP, triggered the neutrophil respiratory burst through different receptors, which led to the production of different amounts of ROS, as illustrated in Fig. 1. It was observed that the ranking orders of neutrophil CL responses to the stimuli were OZ > PMA > fMLP > HBSS (unstimulated

Discussion

Atherosclerosis is an important pathologic manifestation underlying cardiovascular diseases, the leading cause of mortality and morbidity in developed countries. It has been classified as an inflammatory disease, since the presence of humoral and cellular components of the immune response have been detected within the atherosclerotic lesion. It has been suggested that increased production and release of activated leukocyte-derived products, such as ROS, enzymes and pro-inflammatory mediators

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, for financial support (Grants # 130777/2005-6 and 474614/2003-4).

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