Inhibitory effects of areca nut extract on expression of complement receptors and fc receptors in human neutrophils.

BACKGROUND: Chewing of areca quid increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the phagocytic activity of human neutrophils. This in vitro study investigates the effects of ANE on complement- and antibody-opsonized phagocytosis by neutrophils. Expression of complement receptors, Fc receptors, and F-actin in ANE-treated neutrophils is also analyzed. METHODS: The viability of ANE-treated neutrophils was determined using the propidium iodide staining method. The possible effects of ANE on the expression of complement receptors and Fc receptors were examined using an immunofluorescence staining method followed by flow cytometry and confocal laser scanning microscopy. The phagocytic activity of neutrophils against complement or immunoglobulin (Ig)G-opsonized fluorescent beads was analyzed using flow cytometry. Expression of F-actin was determined using confocal laser scanning microscopy. RESULTS: ANE significantly inhibited the production of complement receptors (CR1, CR3, and CR4) and Fc receptors (FcγRII and FcγRIII) in a concentration-dependent manner. Treatment of neutrophils with ANE significantly impaired their ability to phagocytose fluorescent beads. ANE also inhibited phagocytosis of fluorescent beads that were opsonized by complement or IgG. Moreover, expression of F-actin was inhibited after ANE treatment. CONCLUSIONS: ANE inhibits the complement- and IgG-mediated neutrophil phagocytosis that may result from reduction of the expression of complement receptors, Fc receptors, and F-actin formation after ANE treatment. The findings suggest that areca nut chewing may jeopardize the defensive functions of neutrophils and affect periodontal health.

Effects of areca nut extract on lipopolysaccharides-enhanced adhesion and migration of human mononuclear leukocytes.

BACKGROUND: Areca chewers have a higher prevalence of periodontitis than non-chewers. Cell adhesion and movement (migration) are important for leukocyte recruitment to inflammation sites. This study investigates the effects of areca nut extract (ANE) on the adhesion and migration abilities of the human immune cells, peripheral blood mononuclear cells (PBMCs). The combined effects of nicotine and lipopolysaccharides (LPS) were also analyzed. METHODS: Purified PBMCs obtained from healthy adults were treated with ANE, nicotine, and/or LPS. Cell adhesion ability was examined using fibronectin-coated microslides, Liu stain, and light microscopy. Cell migration ability was evaluated using the transwell system followed by staining and fluorescence microscopy. Statistical difference was analyzed using the Mann-Whitney U test. RESULTS: When compared with the media-treated control samples, PBMCs treated with ANE for 4 hours showed a significant reduction of the adherent cells on the microslides. Interestingly, LPS treatment increased cell adhesion, which could be reduced by simultaneous ANE plus nicotine treatment. The chemotactic migration of PBMCs was reduced by ANE treatment for 1, 4, or 24 hours in a dose-dependent manner. LPS treatment increased PBMC migration, which could be reduced by simultaneous treatment with ANE or with ANE plus nicotine. CONCLUSIONS: ANE reduced the adhesion and migration abilities of PBMC. ANEs, with or without nicotine, also attenuated the migration of LPS-stimulated PBMCs. The results implicated that the immune cell functions were impaired in areca chewers, which might increase the host susceptibility to oral and periodontal infection.

Areca nut extracts increased the expression of cyclooxygenase-2, prostaglandin E2 and interleukin-1α in human immune cells via oxidative stress.

BACKGROUND AND OBJECTIVES: Areca nut has been identified as a carcinogen. Inflammation reveals a strong link with tumourigenesis. The aim of this study was to investigate the effects of areca nut on the expression of the key pro-inflammatory mediators involved in malignancy, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), interleukin (IL)-1α and nuclear factor-κB (NF-κB), by human immune cells. The role of oxidative stress was also examined. MATERIALS AND METHODS: Human peripheral blood mononuclear cells (PBMCs) were treated with extracts of ripe areca nut (rANE) or tender areca nut (tANE). Expression of pro-inflammatory mediators was assayed using Western blotting, reverse transcription-polymerase chain reaction, competitive enzyme immunoassay or enzyme-linked immunosorbent assay (ELISA). Activity of NF-κB was evaluated using an ELISA-based method. RESULTS: Both rANE and tANE enhanced the expression of COX-2, PGE2 and IL-1α by PBMCs. The secretion of PGE2 was induced by rANE (≤20-40µgml(-1)) and tANE (≤160µgml(-1)) significantly in a dose- and time-dependent manner. However, the above enhancing effects of ANEs could be attenuated by antioxidants. ANEs also increased the nuclear expression of the redox-sensitive factor NF-κB. CONCLUSIONS: The results demonstrate that ANEs induced the expression of pro-inflammatory mediators mainly through the induction of oxidative stress and implicate the possibility of using antioxidants for disease prevention.

Enhancing effects of areca nut extracts on the production of interleukin-6 and interleukin-8 by peripheral blood mononuclear cells.

BACKGROUND: The habit of chewing areca quid (AQ) has been implicated in oral pathogenesis, including periodontal disease. Little is understood about the roles of AQ in the cytokine secretion by immune cells. The study examined the effects of areca nut, the major ingredient of AQ, on the production of interleukin (IL)-6 and IL-8 by peripheral blood mononuclear cells (PBMC), the immunocompetent cells. The possible role of oxidative stress of areca nut was also examined. METHODS: Extracts of ripe areca nut (rANE) and tender areca nut (tANE) were examined for their cytotoxic effects on human PBMC using the trypan blue exclusion test. The production of IL-6 and IL-8 by ANE-treated PBMC was analyzed using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. Effects of an antioxidant, pyrrolidine dithiocarbamate (PDTC), on ANE-induced cytokine secretion were also studied. RESULTS: At the experimental conditions, 20 micro g/ml rANE decreased cell viability significantly, whereas no significant effect of tANE (< or =80 micro g/ml) was observed. Both rANE (< or =20 micro g/ml) and tANE (< or =160 micro g/ml) significantly increased the secretion of IL-6 and IL-8 by PBMC in a dose- and time-dependent manner. The altered mRNA expression of IL-6 by rANE and tANE was also observed. Moreover, the stimulating effects of rANE on cytokine expression in PBMC could be attenuated by PDTC, suggesting that the oxidative stress of rANE may play a role. CONCLUSIONS: Markedly enhancing effects of ANE on PBMC-released inflammatory cytokines might cause a sustained cytokine-rich inflammatory milieu in oral cavity of AQ chewers. These excessive cytokines from ANE-treated immune cells may impair periodontal health.

Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils.

BACKGROUND: Areca quid chewers have a higher prevalence of periodontal disease than non-chewers. Little is known about the influence of areca quid on the immune system. This study was to determine the possible effects of the areca nut on phagocytic activity of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE), fresh and tender areca nut with husk (tANE), a major alkaloid (arecoline), and a phenolic component ([+]-catechin) of areca nut were examined for their effects on cellular viability using trypan blue exclusion assay. The possible effects on the phagocytic activity of neutrophils against a periodontal pathogen, Actinobacillus actinomycetemcomitans ATCC 33384, were determined using flow cytometry and confocal laser scanning microscopy. RESULTS: At the concentrations tested, rANE, tANE, arecoline, and (+)-catechin did not significantly affect viability of neutrophils. However, rANE, tANE, arecoline, and (+)-catechin inhibited the phagocytic activity of neutrophils in a dose-dependent manner. Approximately 50% of the relative phagocytic activity of neutrophils was affected when 50 microg/ml of rANE, 400 microg/ml of tANE, 20,000 microg/ml of arecoline, or 2,500 microg/ml of (+)- catechin was used. Decreased levels of internalized fluorescent bacteria were also demonstrated. However, arecoline or (+)-catechin alone could not be used to explain the inhibitory effects observed for rANE and tANE. CONCLUSIONS: Components of areca nut reduced the uptake of A. actinomycetemcomitans ATCC 33384 by human neutrophils. The inhibition of areca nut on phagocytosis of neutrophils may be one possible mechanism by which the areca nut compromises the periodontal health of areca quid chewers.