The Journal of Nutrition -. Nutritional Immunology ASN Resveratrol Alters Proliferative Responses and Apoptosis in Human Activated B Lymphocytes in Vitro1'2

Susan J. Zunino , * and David H. Storms

USDA, Agricultural Research Service, Western Human Nutrition Research Center, Davis, CA 95616

Abstract We hypothesized that the phytochemicals resveratrol, quercetin, and kaempferol would modulate B lymphocyte proliferation, Ig synthesis, and apoptosis after activation. Peripheral blood mononuclear cells (PBMC) were isolated from 12 healthy adult human volunteers and incubated with pokeweed mitogen plus 0, 2, 5, and 10 j.rmol/L resveratrol, quercetin, or kaempferol. After 6 d, CD19+ B cells were analyzed for proliferation, B cell lymphoma-2 (Bcl-2) expression, and activation of caspase-3 using flow cytometry. After 8 d, cell supernatants were collected and 1gM and lgG were measured by ELISA. Resveratrol at a concentration of 5 jamol/L increased the percentage of CD19+ cells compared with mitogen only-stimulated cells (P < 0.01), and a trend for increased proliferation was observed for cells treated with 0, 2, and 5 emol/L resveratrol (P-trend = 0.01). However, 10 Rmol/L resveratrol inhibited proliferation of B lymphocytes )P< 0.01). Expression of Bcl-2 and caspase-3 activation increased in B cells treated with 10 Rmol/L resveratrol compared with mitogen alone (P < 0.01), and trends for dose-responsive increases in Bcl-2 expression and caspase-3 activation were observed (P-trend < 0.00011. Differences in IgM and lgG production were not observed for PBMC treated with resveratrol. Kaempferol at 10 emol/L slightly inhibited proliferative responses (P< 0.05) but did not affect B cell function or apoptosis. Quercetin did not alter B cell proliferation, function, or apoptosis. These data show that human B lymphocyte proliferation and apoptosis are modified by physiological concentrations of resveratrol and suggest that exposure of human B cells to resveratrol may increase survival by upregulating Bcl-2. J. Nutr. 139: 1603-1608, 2009.

Introduction lymphocytes and production of Ig specific for the antigen Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a plant antibi- presented. Production and secretion of Ig by activated B otic that functions to protect the plant from fungal infection (1). lymphocytes is regulated by antigen interaction with the B cell Resveratrol is present in the skins, seeds, and leaves of grapes receptor, direct cell-to-cell contact with T lymphocytes, and and a narrow range of other foods, including peanuts, blue- production of cytokines by T helper cells (S). T helper type berries, and cranberries. Quercetin and kaempferol are 2 related 1 lymphocytes produce the cytokines interleukin (IL) 3-2 and flavanols found in many fruits and vegetables (2). Resveratrol, interferon-y that promote the generation of CDS+ cytotoxic I quercetin, and kaempferol have shown antiintlammatory activ- cells (9). T helper type 2 cells produce the cytokines I1.-4, IL-5, ities [reviewed in (3,4)]. Resveratrol, quercetin, and kaempferol and IL-lU that promote B cell proliferation and production of Ig can inhibit the activation of the transcription factor nuclear (9,10). factor-KB, an important mediator of inflammatory responses, as Apoptotic cell death is mediated by the sequential activation well as several enzymes involved in signal transduction path- of caspases, and apoptotic cells exhibit a variety of morpholog- ways (1,5,6). Resveratrol and quercetin inhibit the enzyme ical changes characterized by chromatin condensation, DNA cyclooxygenase-2, which is induced by mitogens, cytokines, and fragmentation, membrane blebbing, and formation of apoptotic bacterial lipopolysaccharide and plays a key role in inflamma- bodies that are eventually phagocytosed in vivo (11). Activation tory processes (1,7). of caspase-3 is an early to intermediate event during apoptosis The humoral immune response is mediated by activation of T that is necessary to induce DNA fragmentation and changes in lymphocytes through interaction with antigen-presenting cells. cellular morphology. Caspase-3 mediates the cleavage of the After activation, T lymphocytes promote the activation of B majority of cytoplasmic and nuclear proteins, including those responsible for maintaining cytoskeletal structure (12). The Supported by the USDA Current Research Information System project no. 5306-51 530-01 3-OOD. 2 s Zunino and D. Storms, no conflict of interest. Abbreviations used: APC, allophycocyanin; Bcl-2, B cell lymphoma-2; DMSO, To whom correspondence should be addressed. E-mail: susan,zunino@ars. dimethylsulfoxide; FITC, fluorescein isothiocyanate; IL, interleukin; PBMC, usda gov peripheral blood mononuclear cells; PWM, pokeweed mitogen. 0022-3166/08 $800 © 2009 American Society for Nutrition. Manuscript received January 23, 2009. Initial review completed February 3, 2009. Revision accepted June 4, 2009. 1603 First published online June 23, 2009; doi: 1 0.3945/jn. 1 09.105064. B cell lymphoma-2 (Bcl-2) family of proteins contains both anti- Resveratrol, quercetin, or kaenipferol were added to the appropriate and proapoptotic proteins. Bcl-2 and Bcl-XL are antiapoptotic wells at final concentrations of 2, 5, and 10 emol/L. Control wells for members of the Bcl-2 family of proteins and are expressed in B proliferation contained PWM and 0.1% DMSO (equivalent to DMSO lymphocytes (13,14). Each of these proteins is partly responsible concentration used for resveratrol, quercetin, or kaempferol). Control wells for nonproliferating cells contained 0.5% PBS and 0.1% DMSO. for maintaining viability after B cell activation. The final volume per well was 200 jd.. Cells were incubated for 6 d at Sharma et al. (15) showed resveratrol inhibited proliferation 37°C, 5% CO2. Cells from duplicate wells were pooled and stained with of activated T and B cells isolated from mouse spleens and APC-conjugated anti-CDI9 antibody for 30 min on ice to identify the B decreased production of 1g. However, significant differences lymphocyte population in each sample. Cells were washed once, fixed in were observed only at resveratrol concentrations of 20 rmol/L. 1% para formaldehyde prepared in PBS, and collected on a FACSCanto To date, no research has been performed to define the effects of fluorescence-activated cell sorter using FACSDiva software (Becton resveratrol, quercetin, or kaempferol on activated human B Dickinson). Fifty thousand events were collected for each sample. All lymphocytes. We hypothesized that due to other antiinflamma- analyses of whole cells were performed using appropriate scatter gates to tory activities ascribed to these polyphenolic compounds, they exclude cellular debris and aggregated cells. Proliferation was measured would inhibit activation, survival, and function of human B on CD19+ gated cells using the Proliferation Wizard feature of ModFit LT software )Verity Software House). For each treatment group, lymphocytes. In this study, we collected peripheral blood comparisons were made between the numbers of cells remaining in the mononuclear cells (PBMC) from healthy human volunteers unproliferated state )parent) and the numbers of cells in subsequent and activated these cells with a polyclonal B cell mitogen to generations. Unstimulated CD 19+ cells were used to locate the parent induce proliferation and Ig synthesis in the absence and presence peak for calculation of generations in the PWM-stimulated cells. of physiological concentrations of resveratrol, quercetin, and kaempferol. Proliferative responses, expression of Bcl-2, activa- Bcl-2 expression and caspase-3 activation. PBMC were plated in tion of caspase-3, and production of 1gM and IgG were duplicate and incubated for 6 d at 37°C as described above before measured to determine whether there was suppression of B analysis of Bcl-2 and caspase-3. Cells from duplicate wells were pooled, lymphocyte activation and survival by these polyphenols. washed once, and stained with APC-conjugated anti-CDI9 for 30 mm on ice. Cells were washed then fixed and permeabilized using Cytofix/ CytoPerm solution (Becton Dickinson) according to the manufacturer's protocol. Cells were washed and stained with FITC-conjugated anti- Materials and Methods caspase-3 and PE-conjugated anti-Bcl-2 antibodies for 30 min on ice. Participants and cell preparation. Twelve healthy adult human Cells were washed, refixed in 1% paraformaldehyde, and fluorescence volunteers between the ages of 18 and 60 y (5 male, 7 female) were was measured by flow cytometry. Fifty thousand events were collected recruited to participate in the study. The human study protocol outlining for each sample. All analyses of whole cells were performed using the ethical treatment and protection of human subjects was approved by appropriate scatter gates to exclude cellular debris and aggregated cells. the Institutional Review Board at the University of California, Davis, Bcl-2 expression and caspase-3 activation were measured in B lympho- CA. The protocol and goals of the study were explained to each cytes by gating the CD 19+ population. Isotype controls for the anti-Bcl-2 volunteer and written consent was obtained. Potential volunteers were and anti-caspase-3 antibodies were used to determine marker settings in excluded if they were overtly ill, had disease states affecting immune cell the fluorescence-activated cell sorter data. function (allergy, asthma, autoimmune disorders, AIDS), or were taking doctor-prescribed medications that affected immune cell responsiveness. 1gM and lgG production. PBMC were plated in duplicate as described Blood was collected at -.1000 h into CPT tubes (Becton Dickinson) from above and were incubated for 8 d at 37°C and supernatants were pooled each nonfasting volunteer by venipuncture and PBMC were isolated from duplicate wells. Supernatants were centrifuged at 900 X g for using the manufacturer's protocol. The plasma was discarded and the 10 min to remove cells and debris and aliquots were stored at —20°C. cells were washed once with PBS. Platelets were removed by centrifu- ELISA kits for measuring human 1gM and IgG were purchased from gation at 200 >< g; 10 min before growth medium was added to the cells. Bethyl Laboratories. EUSA were performed according to the manufac- Growth medium consisted of RPMI 1640 )Invitrogen) supplemented turer's protocol. Measurements for each supernatant were performed in with 10% fetal bovine serum (Sigma), 60 mg/L penicillin, 100 mg/L duplicate on a Synergy 2 microplate reader using GenS software )BioTek streptomycin, 0.25 mg/L amphotericin B, I mmol/L sodium pyruvate, Instruments). 2 mmol/L glutamine )Invitrogen), and 50 imol/L 13-mercaptoethanol (Sigma). Statistical analysis. All statistical analyses were performed with GraphPad Software. One-way repeated-measures ANOVA with Dun- Reagents and antibodies. Resveratrol and kaempferol aglycones and nett's post hoc test and linear trend post tests were used to compare the PKH67GL kit were purchased from Sigma. Quercetin aglycone was proliferation, Ig production, Bcl-2 expression, and caspase-3 activation purchased from Calbiochem. Resveratrol, quercetin, and kaempferol in response to different doses of resveratrol, quercetin, or kaempferol. were dissolved in dimethylsulfoxide (DMSO; Sigma) and stored at Values in the text are means ± SEM. Differences were considered —20°C. Pokeweed mitogen )PWM; Sigma) was dissolved in PBS and significant at P < 0.05. stored at a concentration of 1 gIL at —20°C. Fluorescein isothiocyanate )FITC)-conj ugated anti-human activated caspase-3, PE-conjugated anti- human Bcl-2, and allophycocyanin )APC)-conjugated anti-human CD19 Results were purchased from Becton Dickinson. Control antibodies used included FITC-conjugated rabbit IgG )eBioscience), PE-conjugated Differential proliferative response of B lymphocytes to hamster IgGi (Becton Dickinson), and APC-conjugated mouse IgGI resveratrol. PWM is a T cell-dependent mitogen that induces (Becton Dickinson). proliferation and Ig synthesis in B lymphocytes (16). Maximum proliferative response of B lymphocytes to PWM was reported Proliferation assay. Proliferation responses were measured by staining PBMC with PKH67 following the manufacturer's protocol. The to be 6 d (17). Unstimulated and PWM-stimulated human concentration of PWM that induced maximum proliferation of B PBMC were cultured for 6 d in the presence or absence of 2, 5, or lymphocytes was determined by testing different doses. For analysis of 10 emol/L resveratrol, quercetin, or kaempferol. PBMC were the effects of polyphenols on B cell growth, PBMC were plated in stained with fluorochrome-conjugated anti-CD 19 antibody to duplicate at 100,000 cells per well in 96-well, flat-bottomed microtiter identify the B lymphocyte suhpopulations. The mean percent of plates in the presence or absence of PWM )final concentration of 5 mg/L). CD19+ B lymphocytes in the unstimulated PBMC population 1604 Zunino and Storms

was 5.4 ± 0.3%. The proportion of CD19+ B lymphocytes A 100 increased to 15.2 ± 1.4% in PWM-stimulated PBMC treated 90 with 5 LmolIL resveratrol compared with 11.5 ± 1.1% in cells 80 treated with PWM alone (P < 0.01). PKH67 dye was used to ctJ label the cell membranes before treatments, and the subsequent 0 CM 70 loss of fluorescence was used to determine the percent of CDI 9+ 60 cells in each generation compared with unstimulated cells, U) a)U) where a distinct parent population of B cells was present. A a. 50 trend for decreases in the percent of CD19+ lymphocytes a)>< U, 40 remaining in the parent population after PWM stimulation was a) 0 30 observed with 2 and S imol/L resveratrol treatments compared III with cells stimulated with PWM alone (Fig. 1) (P-trend = 0.01). 20 However, resveratrol at a final concentration of 10 mol/L 10 increased the percent of PWM-stimulated CD 19+ B lymphocytes that remained in the parent population compared with PWM U P 2R 5R 1OR control cells (Fig. I) (P < 0.01). Furthermore, treatment with 10 Lmo1/L resveratrol decreased the percent of CD19+ cells in B the 7th and 8th generations compared with the PWM-stimulated 1400 control cells (P < 0.05). Kaempferol at a final concentration of 10 .tmol/L slightly increased the percent of CD19+ cells in the . 1200 U) second generation to 8.9 ± 1.1% compared with 6.5 ± 0.7% in cells stimulated with PWM alone (P < 0.05). Treatments with quercetin did not alter the proliferative responses of PWM- Q) stimulated CD19+ cells compared with cells stimulated with Cn PWM alone. 600 Expression of BcI-2 and activation of caspase-3. Expression 400 of the antiapoptotic protein Bcl-2 and the activation status of the proapoptotic protein caspase-3 were measured in CDI9+ 200 lymphocytes after 6 d of treatment in the presence or absence of PWM, resveratrol, quercetin, or kaempferol. The percent of CD 19+ cells positive for Bcl-2 or activated caspase-3 and the U P 2R 5R 1OR fluorescent intensity of these intracellularly stained proteins FIGURE 2 Expression of Bcl-2 in CD19+ B cells treated with were used to determine the relative levels of Bcl-2 and activated different concentrations of resveratrol. (A) Percent CD19+ cells caspase-3 proteins. The percentage of cells expressing Bcl-2 expressing Bcl-2. (B( Median fluorescence intensity of PE-conjugated increased in PWM-stimulated cells treated with 10 zmol/L anti-Bcl-2 antibody. Values are means ± SEM, n = 12, *Different from resveratrol compared with PWM-treated control cells (Fig. 2A) P, P < 0.01. U, Unstimulated cells; P, PWM-stimulated cells; 2R, 5R, 1OR represent 2, 5, and 10 mol/L resveratrol. (P < 0.01). The expression level of Bcl-2 protein as measured by median fluorescence intensity also increased in cells incubated with 10 zmol/L resveratrol compared with PWM-treated control cells (Fig. 2B) (P < 0.01). Trends for dose-dependent was observed in CD19+ B cells treated with 2, 5, and 10 mol/L increases in both Bcl-2 expression and intensity of Bcl-2 resveratrol (P-trend < 0.0001). The percent of cells that were fluorescence were observed in PWM-stimulated cells treated double positive for Bcl-2 and activated caspase-3 increased in with 2, 5, and 10 mol/L resveratrol (P-trend < 0.0001). The PWM-stimulated cells treated with 10 mol/L resveratrol percent of cells with activated caspase-3 and the median compared with PWM-treated control cells (Fig. 4) (P < 0.01). fluorescence intensity of the caspase-3 signal increased in A trend for dose-responsive increases in the percent of cells that PWM-stimulated cells treated with 10 zmol/L resveratrol were double positive for Bcl-2 and caspase-3 was observed for compared with PWM-treated control cells (Fig. 3A,B) (P < cells treated with 2, 5, and 10 ILmol/L resveratrol (P-trend < 0.01). A trend for dose-dependent increases in the percent of 0.0001) (Fig. 4). Quercetin and kaempferol did not alter Bcl-2 cells with activated caspase-3 and median fluorescence intensity expression or caspase-3 activation in PWM-stimulated CD19+ cells compared with cells treated with PWM alone.

50 - iJControl

U) M 2MResv Ig synthesis. Ig production was measured after 8 d in 40- * — 5 iM Rosy supernatants of unstimulated and PWM-stimulated PBMC in 0 I 10 iM Resv 30 -] the absence or presence of resveratrol, quercetin, or kaempferol. 1gM and IgG synthesis did not differ between PWM-stimulated 20 0 1 control cells and PWM-stimulated PBMC treated with resver- e 10.1 II ii - * atrol, quercetin, or kaernpferol. 0 rf, I r,.a I F/p I V/, r/. - I I-p I P G2 G3 G4 G5 G6 G7 G8 FIGURE 1 Proliferation of CD19+ B cells incubated with different Discussion concentrations of resveratrol for 8 generations. Values are means ± SEM, n = 12. Different from control at that generation, P < 0.05. P, In this study, proliferation, apoptosis, and function of activated Parent nonproliferating cells; G2—G8 represent cell generations 2-8. human B lymphocytes were analyzed in response to physiolog-

Human B lymphocyte response to resveratrol 1605 A 100 concentration of S mol/L, resveratrol increased the overall number of B lymphocytes after 6 d of stimulation with PWM. 90 Furthermore, there was a trend toward a reduction of B cells in d U) 80 the parent population in the presence of 2 and S mol/L resveratrot, suggesting an increase in proliferative response with coU) 70 0 the lower resveratrol concentrations. However, 10 mol/L a) 60 resveratrol clearly decreased proliferation of B lymphocytes as cu > 50 indicated by the increased number of cells remaining in the 0 (13 parent population and reduction of cells in the 7th and 8th 40 generations compared with PWM-stimulated control cells.

Cd) 30 Treatment of activated human B cells with resveratrol a) increased the expression of Bcl-2, an important antiapoptotic 0 20 protein expressed in activated B and T cells. The fluorescence 10 intensity of anti-Bcl-2 antibody was used to indicate relative amounts of Bcl-2 molecules per cell compared with control cells. U P 2R SR 1OR Both the fluorescence intensity and the percentage of CD19+ cells with Bcl-2 increased after resveratrol treatment. It was B 1000 shown that B and T cells that overexpressed Bcl-2 had delayed cell cycle entry that resulted in increased survival (18,19). 800 Delayed cell cycle entry induced by Bc1-2 was associated with increased expression of the cell cycle inhibitor p27Kipl as well as the retinoblastoma family member p130 (20,21). Further- more, inhibition of cell cycle entry was linked to tyrosine residue 28 in the BH4 domain of Bcl-2 that is independent of the protein's antiapoptotic functions (22). Human CD19+ B cells j treated with 10 mol/L resveratrol had a significant increase in the expression levels of Bcl-2 and a larger percentage of cells remained in the nonproliferating parent population compared 200 with the PWM-stimulated control cells. We also observed a dose-responsive trend toward increased Bcl-2 expression. It is unclear why this trend existed considering the apparent increase U P 2R 5R 1OR in proliferative response in cells treated with 2 and S j.mol/L FIGURE 3 Expression of activated caspase-3 in 0D19+ B cells resveratrol. However, B cells treated with S tmol/L resveratrol treated with different concentrations of resveratrol. (A) Percent CD19+ did have an overall increase in the number of B cells compared cells with activated caspase-3. (B) Median fluorescence intensity of with the other treatment groups, which may argue for increased FITC-conjugated anti-active caspase-3 antibody. Values are means ± survival after activation when cells are exposed to lower SEM, n = 12. Different from F, P < 0.01. U, Unstimulated cells; F, resveratrol concentrations. An increase in activation of cas- PWM-stimulated cells; 2R, 5R, 1 O represent 2, 5, and 10 zmol/L pase-3 was also observed in B cells treated with 10 mol/L resveratrol. resveratrol and the increased caspase-3 activation was observed

ical concentrations of the polyphenols resveratrol, quercetin, and kaempferol. To our knowledge, this is the first report 100 describing the effects of these polyphenols on activated human B 90 lymphocyte proliferation and function. We showed that U) resveratrol modulated B lymphocyte proliferation and apoptosis a) 80 but did not affect Ig synthesis. Quercetin and kaempferol did not 0 + 70 substantially affect B lymphocyte proliferative or apoptotic C') responses, nor did they affect Ig synthesis. Sharma et at. (15) U) 60 (13 described the activity of resveratrol at doses of 1, 5, 10, and 0 U) 50 20 /Lmol/L on the in vitro responses of activated mouse B cells 0(13 and the synthesis of Ig after stimulation with lipopolysaccharide. + 40 (.J

In these studies with mouse splenocyte cultures, the authors 0 30 observed significant inhibition of T and B cell proliferation, Co reduced IL-4 and interferon-y synthesis, increased IL-10 secre- 20 tion, and decreased production of IgGi and IgG2a at a dose of 10 20 AmollL resveratrol. It should be noted that the authors claimed a dose-dependent response for the effect of resveratrol U P 2R 5R 1OR on cytokine synthesis and proliferative response. However, no FIGURE 4 The percent of CD19+ B cells that are double positive for trend analyses were performed to substantiate these observa- Bc)-2 and activated caspase-3. Values are means ± SEM, n = 12. tions. In our study, a differential proliferative response in the *Different from P, P < 0.01. U, Unstimu)ated cells; P, PWM- activated human CD19+ B cell population was observed with 2 stimulated cells; 2R, 5R, 1OR represent 2, 5, and 10 j.imol/L and 5 mol/L compared with 10 tmol/L resveratrol. At a resveratrol. 1606 Zunino and Storms in B lymphocytes with upregulated Bcl-2. Activated B cells are Literature Cited normally recruited to the germinal centers where hypermutation of the Ig gene creates B cell populations with increased specificity I. Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, 1akada Y. Role of resveratrol in prevention and therapy of cancer: for antigen (23). B cells producing low affinity antibodies in the preclinical and clinical studies. Anticancer Res. 2004;24:2783-840. germinal center, as well as those in a cell culture system, undergo 2. Bravo L. Polyphenols: chemistry, dietary sources, metabolism and apoptosis and this apoptotic death can be blocked by Bcl-2 (24). nutritional significance. Nutr Rev. 1 998;56:3 17-33. Taken together, these data suggest that resveratrol may promote 3. Das 5, Das DK. 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1608 Zunino and Storms