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Annals of Clinical & Laboratory Science 33:226-231 (2003)
© 2003 Association of Clinical Scientists

Effects of Resveratrol on Lymphocyte Proliferation and Cytokine Release

Paolo Boscolo1, Antonella del Signore2, Enrico Sabbioni3, Mario Di Gioacchino1, Luca Di Giampaolo1, Marcella Reale4, Pio Conti4, Roberto Paganelli1 and Mario Giaccio2
1 Department of Medicine and Science of Aging, 2 Department of Sciences, and 3 Department of Oncology and Neurosciences, University G. D’Annunzio, Chieti and Pescara, Italy; 4 European Commission, Institute for Health and Consumer Protection, ECVAM Unit, Ispra, Italy.

Address correspondence to Professor Paolo Boscolo, Section of Occupational Medicine, Allergy, and Clinical Immunology, University G. D’Annunzio, Faculty of Medicine and Surgery, via dei Vestini, 66013 Chieti Scalo, Italy; tel and fax 39 087 135 6704; e-mail boscolo{at}unich.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Resveratrol, synthesized in dietary plants and contained in wine, has been reported to play a beneficial role in certain cardiovascular regulatory mechanisms and to inhibit carcinogenesis by activating immune and inflammatory responses and apoptosis. The object of this study was to elucidate the "in vitro" effects of different concentrations of resveratol (10-4, 10-5, and 10-7 M) on human peripheral blood mononuclear cell (PBMC) proliferation and cytokine release. Spontaneous PBMC proliferation was unaffected by resvetratrol, while the compound at 10-4 M inhibited (69 %) the PHA-stimulated PBMC proliferation. The proliferation stimulation index (ie, the ratio of PHA-stimulated PBMC proliferation/spontaneous PBMC proliferation) of cultures containing 10-4 M resveratrol was very low in relation to the control, while the proliferation stimulation index values at 10-5 and 10-7 M were similar and slightly higher (without statistical significance), respectively. At 10-4 M, resveratrol strongly inhibited PHA-stimulated IFN-{gamma} and TNF-{alpha} release from PBMC, but it did not cause inhibition at 10-5 or 10-7 M. The concomitant immune effects of resveratrol on PBMC proliferation and release of IFN-{gamma} and TNF-{alpha} may be explained by an inhibitory effect on transcription factor NF-kappaB. This study suggests that resveratrol, which is typically present in red wine at about 10-5 M, is unlikely to cause inhibitory immune effects. However, a stimulatory effect of low concentrations of resveratrol on the immune system cannot be excluded.

(received 1 February 2003; accepted 6 February 2003)

Keywords: resveratrol, lymphocytes, macrophages, IFN-{gamma}, TNF-{alpha}, immunotoxicity


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It has been demonstrated that polyphenols (and in particular 3,5,4'-trihydroxystilbene, resveratrol) synthesized in dietary plants exert several biological activities [1,2]. Resveratrol is produced by some spermatophytes and is found in grapes, wines, Polygonum roots, peanuts, and berries [1–4]. Four isomeric forms of resveratrol have been identified: trans- and cis-resveratrol and trans- and cis-ß-D-glucopyranoside; these isomeric forms constitute the "total stilbene." Trans-resveratrol and trans- and cis-ß-D-glucopyranoside are present in grapes, whereas all 4 forms are contained in wines. Trans-resveratrol was first detected in grapevines (Vitis vinifera) in 1976 by Langcake and Pryce [3], and its presence was explained as a response to fungal infection, mainly Botrytis cinerea. In 1992 Siemann and Creasy [4] suggested that this compound might be a biologically active constituent of red wine, possibly beneficial for human health. Since then, the content of resveratrol in wines and its properties have been extensively investigated. Recent articles report high variability in resveratrol concentrations depending on wine processing [5], climatic factors that affect grape cultivation [6], yeasts used in the fermentative processes [7], and various other factors [8].

Kuhnle et al [9] demonstrated that resveratrol, which passes through enterocytes of the jejunum and ileum, is not metabolised. During passage through enterocytes, 96.5% of absorbed resveratrol is conjugated with glucuronic acid. Glycosidase activity that converts glucosides to aglycones is exerted during the digestive phase. It was suggested that all of the isoforms (constituting the "total stilbene") are transformed to resveratrol in the digestive tract by this activity; therefore, resveratrol may act "in vivo" as the only biologically active form.

The resveratrol content of red wine (approximately 10-5 M [6]) has been reported to afford cardiovascular protection and to reduce atherosclerosis by various mechanisms [10,11]. These include modulation of lipid turnover, production of eicosanoids, oxidation of lipoproteins, and reduction of platelet adhesion [12,13]. Moreover, resveratrol was reported to inhibit angiotensin-II-induced hypertrophy of vascular smooth cells [14].

Resveratrol has also been reported to be involved in anti-inflammatory and anticancer mechanisms [15]. It apparently affects different stages of carcinogenesis in murine mammary epithelial cells [16]; it limits the ability of human colon carcinoma cells to form colonies through both pro-apoptotic and non-apoptotic mechanisms [17].

Resveratrol has been reported to reduce the risk of cardiovascular and tumoral diseases by acting on the mechanisms that regulate the expression of growth factors and cytokines such as transcription factor NF-kappaB [2,16]. Studies on immune effects of resveratrol have been performed. The compound was shown to inhibit the proliferation and IL-2 and interferon (IFN)-{gamma} production of splenic lymphocytes, the activity of cytotoxic T lymphocytes, the activity of lymphokine-activated killer cells, as well as the production of TNF-{alpha} and IL-12 by peritoneal macrophages [18] . Resveratrol was also reported to exert "in vitro" biphasic immune actions, ie, inhibition at high concentration of cytotoxic T lymphocyte and NK cell activities and the production of IFN-{gamma}, IL-2, and IL-4 by T lymphocytes, and stimulatory immune actions at low concentrations [19].

The object of this study was to elucidate the "in vitro" immune effects of different concentrations of resveratrol on peripheral blood mononuclear cell (PBMC) proliferation and cytokine release.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Resveratrol.  Resveratrol solution (10-3 M) was prepared by diluting 22.82 mg of trans-resveratrol (Sigma Chemical Co, Milan, Italy) in 100 ml of distilled water containing 10% (v/v) of ethanol. This solution was diluted to 10-4, 10-5, and 10-7 M in cultures containing ethanol concentrations from 1 to 0.001%.

Isolation of human PBMC.  Nine healthy men (mean age 34 yr, range 24–58 yr) were recruited for the study. They were unaffected by disease and did not use any drugs. Fasting venous blood samples were obtained from each subject at 8 am. Human PBMC were purified from EDTA-treated whole blood by Ficoll-Hypaque (BioSpa, Milan, Italy) density gradient centrifugation (20 min at 400 x g). After three washings, PBMC were resuspended in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma). This mixture is designated as "complete medium."

Cell proliferation.  PBMC were suspended at 106 cells/ml in complete medium. Aliquots (100 µl) of cell suspension were placed in each well of a standard 96-well microtiter plate (Falcon, St. Louis, MO). The cells were incubated for 78 hr at 37°C in a humidified atmosphere with 5% CO2 under the following conditions:

  1. without addition of phytohemagglutinin (PHA) and resveratrol (control sample),
  2. stimulation by 20 µg/ml PHA without resveratrol (control sample),
  3. as reported in (a) and (b) with the addition of 0.1 ml of ethanol (1%, v/v), and
  4. containing 10-4, 10-5, or 10-7 M resveratrol with and without PHA stimulation.

Quantification of cell proliferation.  Proliferation was evaluated using the BrdU cell proliferation assay (Oncogene Research Products, Darmstadt, FRG). During the final 24 hr of culture, BrdU was added to wells of the microtiter plate. Cells were fixed and permeabilized and DNA was denaturated by treatment for 30 min at room temperature with fixative/denaturing solution. Anti-BrdU monoclonal antibody was pipetted into the wells and allowed to incubate for 1 hr. Unbound antibody was washed away and horseradish peroxidase-coniugated goat anti-mouse antibody was added for 30 min at room temperature. Contents of the wells were removed by inverting over a sink and tapping on paper towels. Chromogenic substrate solution, tetramethyl-benzidine (TMB), was added to each well and incubated in the dark at room temperature for 15 min. Stop solution was added to each well in the same sequence as the previous addition of substrate solution. All of the reagents were provided with the kit and used in accordance with the manufacturer’s instructions. Experiments were performed in triplicate. The absorbance of the contents of each well was measured using a spectrophotometric microtiter plate reader at dual wavelengths (450 and 540 nm). The color intensity was proportional to the amount of incorporated BrdU in the cells and thus to the degree of cell proliferation

Production and measurement of cytokines.  Cultures were set up in 24-well Costar plastic microtiter plates (well volume, 1 ml), using 0.8 ml of PBMC (containing 106 cells) in complete medium under the following conditions:

  1. without addition of PHA or resveratrol (control),
  2. stimulation by 10 µg/ml PHA without resveratrol (control),
  3. as reported in (a) and (b) with addition of 0.1 ml of ethanol (10% v/v), as controls for the cultures containing 10-4 M resveratrol and 1% of ethanol,
  4. stimulation by 10 µg/ml PHA in presence of 10-4, 10-5, or 10-7 M resveratrol.

The cultures were incubated at 37°C in humidified athmosphere with 5% CO2 for 48 hr; cells were checked for viability by trypan blue dye exclusion using an inverted Leica microscope. Supernatants were collected and stored at -70°C in aliquots until analysis. Interferon (IFN)-{gamma}, and tumor necrosis factor (TNF)-{alpha} levels in the culture supernatants were determined by Quantikine ELISA kits (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions.

Statistical analysis.  Statistical analyses were performed with Statistica software (release 4.5). The Kolmogorov-Smirnov test was used to evaluate the data distributions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Kolmogorov-Smirnov test showed that most of the experimental data had non-parametric distributions. In particular, values for cytokine release conformed more to a parametric distribution when they were expressed as a percent of the corresponding controls, rather than as units (pg/ml).

Spontaneous and PHA-stimulated PBMC proliferation, as well as IFN-{gamma} and TNF-{alpha} release from PBMC, were not influenced by the 1% ethanol content of the cultures.

Table 1Go and Fig. 1Go show data for spontaneous and PHA-stimulated PBMC proliferation; Fig. 2Go shows data for PHA-stimulated release of IFN-{gamma} and TNF-{alpha} from PBMC. The following obsevations were made:


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  		Table 1. Spontaneous or PHA-stimulated PBMC proliferation without (control) or with 10-4, 10-5, and 10-7 M resveratrol. Absorbance data (mean ± SD) are expressed as units and as ratios in relation to the control.
 


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  		Fig. 1. Blastogenesis stimulation index (SI = ratio of PHA-stimulated/spontaneous proliferation) of PBMC without (control) or with 10-4, 10-5, and 10-7 M resveratrol. Values are expressed as mean ± SD.

• Significant difference vs control (p <0.001) by Mann-Whitney U test

 


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  		Fig. 2. IFN-{gamma} and TNF-{alpha} in supernatants of PHA-stimulated PBMC without (control) or with 10-4, 10-5, and 10-7 M resveratrol. Values are expressed as ratio vs the control (with/without resveratrol) (mean ± SD).

•Significant difference vs control (p <0.001) by Mann-Whitney U test

 
The spontaneous PBMC proliferation assay without resveratrol (baseline profile) shows an absorbance (170 ± 29 units) that increases to 1633 ± 214 units in PHA-stimulated cultures (mean ± SD; Table 1Go). Thus, an increase of 9–10 fold is the control response to PHA.

Spontaneous PBMC proliferation is not modified at concentrations of 10-4, 10-5, or 10-7 M resvetratrol (Table 1Go). Addition of 10-4 M resveratrol leads to inhibition of 69.4% of the proliferation of the PHA-stimulated PBMC, while exposure of PBMC to 10-5 or 10-7 M resveratrol does not affect the PHA-induced proliferation (Table 1Go).

The stimulation index (SI) of blastogenesis (ratio of PHA-stimulated PBMC proliferation/spontaneous PBMC proliferation) of control cultures is 9.46 ± 1.25, while for those containing 10-4 M resvetratrol the SI is 2.69 ± 0.81 (p <0.001) (Fig. 1Go); there is no effect at 10-5 M resveratrol. On the other hand, the SI observed at 10-7 M resveratrol is 10.72 ± 0.99, which is slightly higher (but without statistical significance) in relation to the control (Fig. 1Go).

Spontaneous IFN-{gamma} release from PBMC was close to the detection limit (7 ± 6 pg/ml), while the PHA-stimulated release was 1205 ± 305 pg/ml; spontaneous TNF-{alpha} release from PBMC was 104 ± 63 pg/ml and the PHA-stimulated release was 2206 ± 551 pg/ml.

At 10-4 M, resveratrol almost completely inhibited PHA-stimulated release of IFN-{gamma} from PBMC (12.1 ± 10.4% of the control) (Fig. 2Go) and that of TNF-{alpha} (21.6 ± 14.9% of the control), while the exposure to 10-5 and 10-7 M resveratrol did not exert effects (Fig. 2Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that 10-4 M resveratrol strongly inhibits PHA-stimulated PBMC proliferation and cytokine release, while it does not exert these effects at 10-5 M. The content of trans-resveratrol in wines is about 1–2 mg/L (about 0.5 x 10-5 M), whereas the content of "total stilbene" (trans- and cis-resveratrol and trans- and cis-ß-D-glucopyranoside) is typically in the range of 5–10 mg/L (about 2–5 x 10-5 M) [6]. It has been suggested that "total stilbene" of red wine is transformed to resveratrol in the intestinal tract before it is absorbed as glucuronide [9]. Therefore, it seems unlikely that low intake of wine would exert immune effects, while high intake of wine (>1 L/day) may inhibit immune functions related to PBMC proliferation and cytokine production.

PHA-stimulated PBMC proliferation needs the activation of several metabolic functions, including IL-2 production and release [20]. It is known that TNF-{alpha} is produced by a variety of cell types including T and B lymphocytes, NK cells, macrophages, astrocytes, and dendritic cells (21). This cytokine exerts several important biological activities including those involved in septic shock and chronic diseases such as rheumatoid arthritis; it is also shown to be cytotoxic for many tumor cells, inducing apoptosis [22]. On the other hand, IFN-{gamma} production (mainly due to T lymphocytes) may be considered the expression of a Th-1 type cell response [23]. In this regard, the immune effects of resveratrol, concomitantly acting on PBMC proliferation and production and release of IFN-{gamma} and TNF-{alpha}, may be explained by its inhibitory effect on the transcription factor NF-kappaB [2,16]. This trancription factor is strongly linked to immune and inflammatory responses, regulation of cell proliferation, and apoptosis. For this reason, involvement of resveratrol in anti-cancer mechanisms may be considered; possible use of resveratrol as a chemo-therapeutic agent may merit investigation in accordance with the results and suggestions of other authors [15–17].

Resveratrol may interact with metals and chemical compounds present in wine or constituents of the diet. In this regard, it was found that ethanol, present in wine at concentrations from 8 to 14%, potentiated the immune effects of resveratrol on murine macrophages [24].

In conclusion, this study demonstrates that resveratrol inhibits immune functions only at high concentrations. Moreover, this compound does not exert effects at low concentrations that might be likely to reduce the risks of degenerative [10–14] and neoplastic [15–17] diseases. On the other hand, it cannot be excluded that resveratrol, at low concentrations, might act on transcription factors (eg, NF-kappaB), and concomitantly affect immune, inflammatory, and apoptotic mechanisms [2–16)].


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  4. Siemann EH, Creasy LL. Concentration of the phyto-alexin resveratrol in wine. Am J Enol Vitic 1992;43:49–52.[Abstract/Free Full Text]
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  6. Franco MA, Coloru GC, Manca G, Giaccio M, Del Signore A. Level of cis- and trans-resveratrol and their glucosides as a function of different cultivar of red wines. J Commodity Sci 2001;40:167–188.
  7. Franco MA, Coloru GC, Del Caro A, Emonti G, Farris GA, Manca G, Massa TG, Pinna G. Variability of resveratrol (3,5,4'–trihydroxystilbene) content in relation to the fermentation processes by Saccharomyces cerevisiae strains. Eur Food Res Technol 2002;214:221–225.
  8. Goldberg DM. The role of wine phenolics in disease prevention.Riv Vitic Enol 1998;LI:15–31.
  9. Kuhnle G, Spencer JP, Chowrimootoo G, Schroeter H, Debman ES, Srai SK, Rice-Evans C, Hahn U. Resveratrol is absorbed in the small intestine as resveratrol glucuronide. Biochem Biophys Res Comm 2000;272:212–217.[Medline]
  10. Hung LM, Chen JK, Huang SS, Lee RS, Su MJ. Cardio-protective effect of resveratrol, a natural antioxidant derived from grapes. Cardiovascular Res 2000;47:549–555.[Abstract/Free Full Text]
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