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Anti-inflammatory Activity of a High-molecular-weight Cranberry Fraction on Macrophages Stimulated by Lipopolysaccharides from Periodontopathogens

Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec City, Québec, Canada G1K 7P4

^* corresponding author, Daniel.Grenier{at}greb.ulaval.ca

	ABSTRACT

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Periodontitis is a chronic inflammatory disease affecting oral tissues. The continuous, high production of cytokines by host cells triggered by periodontopathogens is thought to be responsible for the destruction of tooth-supporting tissues. Macrophages play a critical role in this host inflammatory response to periodontopathogens. The aim of this study was to investigate the effect of non-dialyzable material prepared from cranberry juice concentrate on the pro-inflammatory cytokine response of macrophages induced by lipopolysaccharides (LPS) from Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum subsp. nucleatum, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Escherichia coli. Interleukin-1 beta (IL-1ß), IL-6, IL-8, tumor necrosis factor alpha (TNF-), and Regulated on Activation Normal T-cell Expressed and Secreted (RANTES) production by macrophages treated with the cranberry fraction prior to stimulation by LPS was evaluated by ELISA. Our results clearly indicate that the cranberry fraction was a potent inhibitor of the pro-inflammatory cytokine and chemokine responses induced by LPS. This suggests that cranberry constituents may offer perspectives for the development of a new therapeutic approach to the prevention and treatment of periodontitis.

KEY WORDS: cranberry • periodontopathogen • macrophage • anti-inflammatory • cytokine

	INTRODUCTION

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Periodontal diseases are a group of inflammatory disorders that lead to the destruction of tooth-supporting tissues and are caused by a specific group of Gram-negative anaerobic bacteria, including Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia (Haffajee and Socransky, 1994). The host response to these bacteria and their products is a critical determinant in the initiation and progression of periodontitis. More specifically, lipopolysaccharides (LPS) of Gram-negative bacteria are potent inducers of pro-inflammatory mediators and can initiate numerous host-mediated destructive processes (Henderson et al., 1996). Monocytes and macrophages, which are found in higher numbers in active periodontal lesions than in inactive sites (Zappa et al., 1991), play an important role in the host inflammatory response to periodontopathogens (Kornman et al., 1997). The continuous, high secretion of various cytokines—including interleukin-1ß (IL-1ß), IL-6, IL-8, and tumor necrosis factor alpha (TNF-) by host cells following stimulation by periodontopathogens—modulates periodontal tissue destruction (Okada and Murakami, 1998). Active compounds endowed with a capacity to modulate the host inflammatory response are now receiving considerable attention, since they may be potential new therapeutic agents for the treatment of periodontal diseases (Paquette and Williams, 2000).

The cranberry is a native North American fruit with various beneficial properties for human health, such as the inhibition of human cancer cell line proliferation (Ferguson et al., 2004; Seeram et al., 2004) and the prevention of adherence of urinary tract infectious agents (Raz et al., 2004). In the area of dental research, it has been reported that a high-molecular-weight fraction prepared from cranberry juice inhibits the co-aggregation of many oral bacteria (Weiss et al., 1998) and affects dental biofilm formation (Steinberg et al., 2004; Yamanaka et al., 2004). In addition, this cranberry fraction reduces mutans streptococci levels in saliva, inhibits in vitro adhesion of Streptococcus sobrinus to hydroxyapatite (Weiss et al., 2004), and promotes S. sobrinus desorption from artificial biofilms (Steinberg et al., 2005).

In this study, we hypothesized that the cranberry may have a beneficial effect in periodontitis by exerting an anti-inflammatory effect. Therefore, we investigated the effect of a high-molecular-weight cranberry fraction prepared from juice concentrate on the production by macrophages of pro-inflammatory cytokines and chemokines associated with periodontitis. More specifically, the cytokine and chemokine responses of macrophages were induced by LPS prepared from Escherichia coli and from the major periodontopathogens: Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum subsp. nucleatum, P. gingivalis, T. denticola, and T. forsythia.

	MATERIALS & METHODS

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Cranberry Fraction
Concentrated juice from the American cranberry Vaccinium macrocarpon was kindly provided by Ocean Spray Cranberries, Inc. (Lakeville-Middleboro, MA, USA). The juice was exhaustively dialyzed (5 days) in 14,000 MW cut-off dialysis bags at 4°C against distilled water and then lyophilized. The non-dialyzable material was considered as fraction 1. Undialyzed concentrated juice was also lyophilized and represented fraction 2. The cranberry powders were dissolved in distilled water prior to use. Chemical analyses of fraction 1 were realized by Robin Roderick (Ocean Spray Cranberries, Inc.) and revealed that this fraction is devoid of sugars and acids and contains 0.35% of anthocyanins (0.055% of cyanidin-3-galactoside, 0.003% of cyanidin-3-glucoside, 0.069% of cyanidin-3-arabinoside, 0.116% of peonidin-3-galactoside, 0.016% of peonidin-3-glucoside, and 0.086% of peonidin-3-arabinoside) and 65.1% of proanthocyanidins. In addition, commercial epigallocatechin gallate (EGCG; Sigma Chemical Co., St. Louis, MO, USA), a polyphenol isolated from green tea, was used as a positive control (Yang et al., 1998).

LPS Preparation
A. actinomycetemcomitans ATCC 29522, F. nucleatum subsp. nucleatum ATCC 25586, P. gingivalis ATCC 33277, T. denticola ATCC 35405, and T. forsythia ATCC 43037 were grown in their appropriate culture media (Grenier, 1996). LPS were isolated from these bacterial strains, as previously reported (Darveau and Hancock, 1983). This method is based on protein digestion of a whole-cell extract by proteinase K and successive solubilization and precipitation steps. The LPS preparations were freeze-dried and kept at –20°C. The amount of contaminating protein was evaluated with the use of a protein assay kit (Bio-Rad Laboratories, Mississauga, ON, Canada), with bovine serum albumin as a control, and was less than 0.001% in all LPS preparations. T. denticola possesses a lipooligosaccharide (although it will be called LPS in the present paper) that has properties rather distinct from those of the classic LPS of the Bacteroides group (Schultz et al., 1998). A standard LPS preparation from E. coli O55:B5 (Sigma Chemical Co.) was also used.

Monocyte and Macrophage Cultures
U937 cells (ATCC CRL-1593.2), a monoblastic leukemia cell line, were cultivated at 37°C in a 5% CO₂ atmosphere in RPMI-1640 medium (HyClone Laboratories, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (RPMI-FBS) and 100 µg/mL of penicillin-streptomycin. Monocytes (2 x 10⁵ cells/mL) were incubated in RPMI-FBS containing 10 ng/mL of phorbol myristic acid (PMA; Sigma) for 48 hrs to induce differentiation into adherent macrophage-like cells, as previously reported (Rovera et al., 1979). Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 hrs prior to use. Adherent macrophages were suspended in RPMI-FBS and centrifuged at 200 x g for 8 min. They were washed and suspended in RPMI with 1% heat-inactivated FBS at a density of 1 x 10⁶ cells/mL and seeded in a six-well plate (2 x 10⁶ cells/well in 2 mL) at 37°C in a 5% CO₂ atmosphere.

Treatment of Macrophages
The macrophages were treated with increasing concentrations of fraction 1, fraction 2, and EGCG, ranging from 10 to 50 µg/mL, and incubated at 37°C in 5% CO₂ for 2 hrs before stimulation with LPS at a final concentration of 1 µg/mL. After a 24-hour incubation (37°C in 5% CO₂), the culture medium supernatants were collected and stored at –20°C until used. Cells incubated in culture medium with or without cranberry fraction or EGCG, but not stimulated with LPS, were used as controls.

Cell Viability
Macrophage viability was evaluated by 0.2% Trypan Blue staining. Cell viability of macrophages was also evaluated by a MTT (3-[4,5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test, according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany).

Determination of Cytokine Production
We used commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) to quantify IL-1ß, IL-6, IL-8, TNF-, and RANTES concentrations in the cell-free culture supernatants, according to the manufacturer’s protocols. The absorbance at 450 nm was read in a microplate reader with the wavelength correction set at 550 nm.

Statistical Analyses
We performed two-way analyses of variance to compare the means of the different conditions. Differences were deemed significant at the 0.05 level (P value). Protected Fisher least-significant differences were used for pairwise comparisons.

	RESULTS

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Effects of Cranberry Fractions on LPS-induced Pro-inflammatory Cytokine Production
To investigate the effects of cranberry fractions on pro-inflammatory cytokine production, we treated macrophages with lyophilized non-dialyzable material of cranberry juice concentrate (fraction 1) or lyophilized cranberry juice concentrate (fraction 2) prior to stimulation with the LPS of major periodontopathogens. To exclude the possibility that cell toxicity due to the cranberry fractions might have been responsible for a decrease in cytokine levels, we evaluated the viability of the macrophages by an MTT test and Trypan blue exclusion. No obvious cytotoxic effects were detected following treatments of macrophages with both fractions, and cell viability was 94% of the untreated controls in all experiments (data not shown).

For IL-1ß, TNF-, and IL-6, the interaction between the two factors LPS and cranberry was significant (P < 0.05), and the results of pairwise comparisons were used. The TNF- and IL-6 responses of the macrophages stimulated by LPS from A. actinomycetemcomitans were significantly reduced by the treatments with fraction 1 (25 and 50 µg/mL) and EGCG (10 µg/mL) (Figs. 1A, 1B). This effect was not observed when the macrophages were treated with fraction 2. Among the other LPS tested, the LPS from F. nucleatum subsp. nucleatum and E. coli induced a TNF- response, whereas an IL-6 response was induced only by the LPS of F. nucleatum subsp. nucleatum (Table). Fraction 1 at a final concentration of 50 µg/mL inhibited the TNF- and IL-6 responses of macrophages induced by the LPS of F. nucleatum subsp. nucleatum, as well as the TNF- response induced by the LPS of E. coli.

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of treating macrophages with fraction 1 (non-dialyzable material of cranberry juice), fraction 2 (cranberry juice), and epigallocatechin gallate (EGCG) on the secretion of IL-6 (A), TNF- (B), and IL-1ß (C) induced by LPS (1 µg/mL) of A. actinomycetemcomitans ATCC 29522 for 24 hrs. Macrophages were treated with cranberry fractions, or EGCG, for 2 hrs prior to lipopolysaccharides (LPS) stimulation. Cytokine secretion was assessed by ELISA. The data are the means ± standard deviations of triplicate assays for three independent experiments. *P value of < 0.05 compared with untreated control.

View this table: [in this window] [in a new window]   Table. Effect of the Cranberry Non-dialyzable Material (fraction 1) on the Secretion by Macrophagesa of IL-1ß, TNF-, IL-6, IL-8, and RANTES Induced by LPS (1 µg/mL) of F. nucleatum subsp. nucleatum ATCC 25586, P. gingivalis ATCC 33277, T. denticola ATCC 35405, T. forsythia ATCC 43037, and E. coli O55:B5 for 24 hrs

Effects of Cranberry Fractions on LPS-induced Chemokine Production
For IL-8, the interaction between LPS and the cranberry fractions was not significant, and the effects of cranberry fraction 1 were analyzed without discrimination of LPS source. LPS of A. actinomycetemcomitans induced a higher IL-8 response than did the other LPS tested (P < 0.05). Fraction 1 (50 µg/mL) significantly reduced the IL-8 response of macrophages stimulated with LPS (Fig. 2A, Table). Fraction 2 and EGCG had no effect on IL-8 production by LPS-stimulated macrophages.

View larger version (25K): [in this window] [in a new window]   Figure 2. Effect of treating macrophages with fraction 1 (non-dialyzable material of cranberry juice), fraction 2 (cranberry juice), and epigallocatechin gallate (EGCG) on the secretion of IL-8 (A) and RANTES (B) induced by LPS (1 µg/mL) of A. actinomycetemcomitans ATCC 29522 for 24 hrs. Macrophages were treated with cranberry fractions, or EGCG, for 2 hrs before lipopolysaccharides (LPS) stimulation. Cytokine secretion was assessed by ELISA. The data are the means ± standard deviations of triplicate assays for three independent experiments. *P value of < 0.05 compared with untreated control.

	DISCUSSION

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The host inflammatory response to periodontopathogens is considered a major factor causing the local tissue destruction observed in periodontitis. Macrophages participate in the host response induced by periodontopathogens and are the principal target for LPS. To determine whether cranberry extracts can interfere with LPS signaling and reduce the production of pro-inflammatory molecules, we stimulated cranberry-fraction-treated macrophages with the LPS from major periodontopathogens and from E. coli. The cranberry non-dialyzable material significantly reduced LPS-induced pro-inflammatory cytokine and chemokine production.

Cytokines, more particularly IL-1ß, are potential markers of the progression and severity of periodontitis as well as indicators of an appropriate response to treatment (Hou et al., 1995). It has been reported that cytokine synthesis inhibitors can reduce bone resorption in experimental periodontitis in rats (Lima et al., 2004). Moreover, local inhibition of both IL-1 and TNF production in periodontal tissues significantly inhibits the inflammatory response and bone loss in ligature-induced periodontitis in monkeys (Assuma et al., 1998). This suggests that local inhibition of cytokines may be a successful approach for inhibiting bone resorption in periodontitis. In this study, we showed that treating macrophages with the non-dialyzable material of cranberry juice can inhibit LPS-induced IL-1ß, TNF-, and IL-6 production and may thus contribute to reducing the impact of cytokine-mediated host destructive processes in periodontitis.

IL-8 and RANTES are potent chemokines that direct the migration of neutrophils, eosinophils, monocytes, and T_H1 cells to sites of infection (Luster, 1998). Stimulation of chemokine production by periodontopathogens favors the accumulation of leukocytes during active inflammation, which contributes to periodontal tissue destruction. Interestingly, periodontal therapy reduces cell numbers in the infiltrate and the levels of IL-8 and RANTES, suggesting a relationship between these chemokines and periodontal status (Gamonal et al., 2001). The non-dialyzable material of cranberry juice reduced LPS-induced IL-8 and RANTES production by macrophages. In the context of the development of novel therapeutic strategies targeting the control of periodontal inflammatory reactions, these results suggest that this cranberry fraction may help reduce the influx of inflammatory cells at disease sites.

The lyophilized cranberry juice did not show any capacity to inhibit LPS-induced cytokine production by macrophages. This is likely related to the fact that active compounds were concentrated in the non-dialyzable material of cranberry juice. One such group of compounds that showed a 125-fold enrichment was the proanthocyanidins (data not shown). EGCG, the green tea polyphenol used as a positive control, also showed anti-inflammatory activity. This is in agreement with the previously reported capacity of EGCG to inhibit LPS-induced TNF- production by mouse macrophages (Yang et al., 1998). Previous studies have revealed that some plant flavonoids may inhibit the expression of inflammation-related proteins/enzymes by suppressing activation of transcription factors such as nuclear transcription factor-B and activator protein-1 (Kim et al., 2004). Future studies will investigate the cellular mechanisms by which cranberry constituents modulate cytokine expression.

Therapeutic agents that modulate host inflammatory mediators have shown promise for managing adult periodontitis and may be highly useful for individuals with a substantially increased risk for periodontitis (Kornman, 1999). In addition to the previously recognized inhibitory effect of the cranberry non-dialyzable material fraction on the aggregation of oral bacteria and dental biofilm formation (Weiss et al., 1998; Steinberg et al., 2004), we showed that this fraction was a potent inhibitor of the pro-inflammatory cytokine and chemokine responses induced by periodontopathogens and E. coli. This provides promising perspectives for the development of novel host-modulating therapies for adjunctive treatments of periodontitis or other inflammatory diseases by use of the high-molecular-weight constituents from cranberries.

	ACKNOWLEDGMENTS

 
This work was supported by the Cranberry Institute (East Wareham, MA, USA). We thank Robin Roderick and Marge Leahy (Ocean Spray Cranberries, Inc.) for chemical analyses and for providing cranberry juice concentrate.

Received April 25, 2005; Last revision October 14, 2005; Accepted October 26, 2005

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