- Email: [email protected]
Dietary nucleotides and human immune cells. II. Modulation of PBMC growth and cytokine secretion
Nutrition 21 (2005) 1003–1009 www.elsevier.com/locate/nut
Applied nutritional investigation
Dietary nucleotides and human immune cells. II. Modulation of PBMC growth and cytokine secretion Elisabeth Holen, Ph.D.*, Oddvin A. Bjørge, M.D., and Roland Jonsson, Ph.D. Broegelmann Research Laboratory, University of Bergen, Bergen, Norway Manuscript received October 21, 2004; accepted March 24, 2005.
Abstract
Objective: The immune system is dependent on purines and pyrimidines as building blocks for DNA and RNA synthesis to enable rapid cell proliferation and protein synthesis. Emerging evidence suggests that dietary nucleotides optimize immune function. We investigated whether growth and function of human immune cells were affected by an exogenous source of nucleotides during specific antigen challenge. Methods: Peripheral blood mononuclear cells from healthy individuals (n ⫽ 10) were stimulated with influenza virus antigen and DNA-Na⫹ from fish soft roe, RNA from bakers yeast (Saccharomyces cerevisiae), 2=deoxyadenosine 5=-monophosphate sodium, 2=deoxycytidine 5=-monophosphate sodium, 2=deoxyguanosine 5=-monophosphate sodium, or 2=deoxyuridine 5=-monophosphate disodium. Growth effects were ascertained by measuring the amount of tritium-labeled Thymidine 5=-monophosphate sodium incorporated into cell DNA. Cell function was measured by detection of interferon-␥ (IFN-␥), tumor necrosis factor-␣, and interleukin-10 production. Results: Specific nucleotide derivatives alone did not affect the growth of healthy peripheral blood mononuclear cells. However, the nucleotide derivatives influenced immune cell growth and cytokine secretion when cocultured with specific antigen. DNA, RNA, deoxyadenosine monophosphate, deoxycytidine monophosphate, and deoxyuridine monophosphate increased influenza virus antigeninduced immune cell proliferation. In contrast, deoxyadenosine monophosphate and thymosine monophosphate inhibited the antigen-induced growth response. RNA and deoxyadenosine monophosphate cocultured with virus antigen significantly increased peripheral blood mononuclear cell secretion of IFN-␥, interleukin-10, and tumor necrosis factor-␣. DNA increased virus antigeninduced immune cell secretion of IFN-␥ only, whereas deoxyuridine monophosphate significantly increased secretion of interleukin-10 only. Deoxyguanosine monophosphate completely inhibited virus-triggered IFN-␥ secretion, whereas thymosine monophosphate did not change the secretion pattern of measured cytokines. Conclusion: Nucleotide derivatives affect growth and function of specific virus antigen-stimulated human immune cells in vitro. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Peripheral blood mononuclear cells; DNA; RNA; Nucleotides; Influenza virus antigen
Introduction The immune system is dependent on adequate nutrition for different vital processes such as energy production, protein synthesis, proliferation, and other specific metabolic
This study was supported by Bjørge Biomarin AS. * Corresponding author. Tel.: ⫹47-55-905-134; fax: ⫹47-55-905-299. E-mail address: [email protected] (E. Holen). 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2005.03.003
pathways. Immune cells require several nutrients to increase their growth rate and function [1,2]. Dietary nucleic acids and nucleotides influence optimal growth and cell function [3,4]. In rodents, nucleotide-free diets decrease cellular and humoral immune responses [5–7] and resistance to bacterial and fungal pathogens [8 –11]. Further, dietary nucleotides can modulate type 1 and type 2 T-helper (Th) cell responses by promoting a shift in the Th1/Th2 balance toward Th1dominant immunity [12–14]. In fish, dietary nucleotides seem to enhance non-specific immune responses [15] and
1004
E. Holden et al. / Nutrition 21 (2005) 1003–1009
vaccine efficacy [16]. In humans, dietary nucleotides increase immune responses to Haemophilia influenzae virus b and diphtheria [17], and polynucleotides significantly increase in vitro antibody production in response to T-cell– dependent antigen [9]. Nucleotides, in addition to being involved in well-known energy-dependent processes, exhibit functions as pluripotent signaling molecules that elicit direct effects on cell functions by purine and pyrimidine receptors but probably also other receptors to induce gene expression. Because dietary nucleotides may exert a wide range of effects depending on nucleotide concentration, structure, and possible receptor affinity, reflected by their target cells, it is necessary to evaluate the effect of various nucleotides on different cell types. In this study we investigated DNA isolated from fish soft roe, RNA from bakers yeast, 2=deoxyadenosine 5=-monophosphate sodium (dAMP-Na⫹), 2=deoxycytidine 5=-monophosphate sodium (dCMP-Na⫹), 2=deoxyguanosine 5=monophosphate sodium (dGMP-Na⫹), 2=deoxyuridine 5=monophosphate disodium (dUMP-Na⫹), and Thymidine 5=monophosphate sodium (TMP-Na⫹) to determine their capacity to stimulate immune cell growth and function and used influenza virus antigen as an immunostimulant in vitro.
Materials and methods DNA and RNA derivatives Sodium salt of DNA from fish soft roe (Bjørge Biomarin AS). Nucleotides dUMP-Na⫹ (D3876), dGMP-Na⫹ (D9500), dCMP-Na⫹ (D7625), dAMP-Na⫹ (D6250), TMP-Na⫹ (T7004) and RNA type III from bakers yeast (R7125) were purchased from Sigma (St. Louis, MO, USA). TMP in this study was used because of unpublished results that indicated effects on growth and immune function in rats fed a TMPsupplemented diet. Initially, all nucleotide derivatives were titrated to find optimal concentrations for cell culture analysis and were estimated from trials that included virus antigen because no proliferation responses were observed with nucleotides and peripheral blood mononuclear cells (PBMCs) alone. To visualize different concentrations of nucleotides, we used 50 g/mL, 1 mg/mL, and 5 mg/mL as suitable concentrations of DNA and RNA for cell culture analysis. Higher concentrations (up to 20 mg/mL) did not essentially change cell growth or function. Nucleotides were used in final concentrations of 50 g/mL and 1 mg/mL.
proliferation response in most PBMCs tested. The virus antigen solution of 1.3 g/mL was the concentration at which most PBMCs showed a significant lower response to virus antigen, compared with the 0.33-g/mL dose, probably due to an earlier onset of cell damage and nonresponsiveness to nucleotide addition. Cells PBMCs from 10 healthy volunteers were isolated from the interface after Ficoll-Paque gradient centrifugation (Pharmacia LKB, Uppsala, Sweden), washed, and resuspended in RMPI-1640 medium supplemented with 10% heat inactivated foetal calf serum and 1% of a 200-mM L-glutamine solution (Gibco BRL, Paisley, UK). Proliferation assay PBMCs (⬎1 ⫻ 105 cells/well) were cultured in 24-well culture clusters (Costar, Cambridge, MA, USA) with or without antigen/DNA/nucleotides and RNA in the concentrations described to a final volume of 2 mL. All reagents were added at initiation of cultures. Five days after initiating cultures, each culture was mixed with a pipette. From each well, 200 L in triplicate was seeded into 96-well cell culture clusters (Costar) and pulsed overnight by adding 20 L of 0.05 mCi/mL [methyl-3H]-thymidine (Amersham, Copenhagen, Denmark). Cultures were harvested into filter mats by using a semiautomatic cell harvester (Skatron, Lier, Norway). Harvested filters and 3 mL of an Ultima Gold liquid scintillation cocktail (Packard Instruments, Downers Grove, IL, USA) were counted in Skatron scintillation vials by a liquid scintillation counter (LKB Wallac 1217, Rackbeta, Bromma, Sweden). Proliferation responses Results are presented as determinations per minute (dpm) means of 10 individual PBMCs. Each PBMC was measured in triplicate as the dpm mean ⫾ standard error of the mean. Unstimulated PBMCs and PBMCs stimulated by an unrelated protein, casein-Na⫹, were included as controls. PBMC supernatants The 24-well PBMC cultures were harvested on day 6 and cells were removed by centrifugation. Cell supernatants were stored at ⫺20°C for cytokine profile analysis. Cytokine analysis
Antigens Influenza virus antigen (A/Beijing/262/95, HxH12W) at final concentrations of 0.33 and 1.3 g/mL of culture was used as the stimulating antigen. Virus antigen diluted to 0.33 g/mL was the concentration that produced the highest
Estimations of interferon-␥ (IFN-␥; DY285, detection limit 15.6 pg/mL), tumor necrosis factor-␣ (TNF-␣; DY210, detection limit 15.6 pg/mL), and interleukin-10 (IL-10; DY217, detection limit 46.9 pg/mL) in culture supernatants of human PBMCs were performed with commer-
E. Holden et al. / Nutrition 21 (2005) 1003–1009
Fig. 1. DNA-Na⫹ from fish soft roe (50 g/mL) and RNA from bakers yeast (50 g/mL or 1 mg/mL) significantly increases Inf-induced immune cell growth. Inf (0.33 g/mL) induced significant proliferation of peripheral blood mononuclear cells compared with control casein (P ⫽ 0.0367), Triplicate measurements of peripheral blood mononuclear cells from 10 healthy subjects were investigated. Results are presented as mean dpm ⫾ standard error of the mean. Differences are significant at P ⬍ 0.05. dpm, determinations per minute; Inf, influenza virus antigen.
cial enzyme-linked immunosorbent assay kits (R&D, London, UK) according to the manufacturer’s instructions. The manufacturer provided the specificity and sensitivity of the kits. Statistics The computer program GrapPad Prism 3.0a (Graph Pad Software, Inc., San Diego, CA, USA) was used for statistical analysis of significant differences. Two-way analysis of variance and t test (to compare each mean with the control mean) were used. P ⬍ 0.05 was considered statistically significant.
1005
Fig. 2. Nucleotides dAMP, dCMP (50 g/mL, 1 mg/mL), and dUMP (50 g/mL) significantly increase Inf-induced immune cell growth. Triplicate measurements of peripheral blood mononuclear cells from 10 healthy subjects were investigated. Growth responses are presented as mean dpm ⫾ standard error of the mean. Differences are significant at P ⬍ 0.05. dAMP, deoxyadenosine monophosphate; dCMP, deoxycytidine monophosphate; dpm, ??; dUMP, deoxyuridine monophosphate; Inf, influenza virus antigen.
in particular, but also 1 mg/mL (except 1 mg/mL of dUMP and DNA), significantly triggered virus-induced PBMC growth. The highest proliferation responses, compared to virus antigen alone (1897 mean dpm) were observed in PBMC cultures in which RNA, dAMP, and dCMP were added with virus antigen (3970-, 4326-, 4086- mean dpm respectively, Figs. 1 and 2). In contrast, dGMP and TMP (at all concentrations tested) showed no significant effect on influenza virus-antigen induced growth responses (Fig. 3).
Results Growth responses DNA-Na⫹ from fish soft roe, RNA from bakers yeast, or mononucleotides did not trigger growth of PBMCs from healthy subjects (n ⫽ 10, P ⬎ 0.05; Fig. 1). Influenza virus antigen (0.33 g/mL) induced significant immune cell proliferation compared with the control casein (P ⫽ 0.0367). In contrast, 1.3 g/mL of virus antigen produced a three-fold lower mean response (Fig. 1) and was not significantly different from the mean of the control group. Interestingly, when cultured with virus antigen, DNA-Na⫹, and RNA (Fig. 1), dAMP, dCMP, and dUMP (Fig. 2) significantly (P ⬍ 0.05) increased virus antigen-induced PBMC proliferation responses. Nucleotide concentrations of 50 g/mL
Fig. 3. Nucleotides dGMP and TMP, when cocultured with Inf, inhibit virus-induced growth of peripheral blood mononuclear cells. Responses are presented as dpm mean ⫾ standard error of the mean (n ⫽ 10, P ⬍ 0.05). dGMP, deoxyguanosine monophosphate; dpm, ??; Inf, influenza virus antigen; TMP, thymosine monophosphate.
1006
E. Holden et al. / Nutrition 21 (2005) 1003–1009
Table 1 DNA-, RNA-, and mononucleotide-induced secretion of TNF-␣, IL-10, and INF-␥ by peripheral blood mononuclear cells from 10 healthy subjects* Culture
TNF-␣ (pg/mL)
IL-10 (pg/mL)
Control DNA 50 g DNA 1 mg DNA 5 mg RNA 50 g RNA 1 mg RNA 5 mg dAMP 50 g dAMP 1 mg dCMP 50 g dCMP 1 mg dGMP 50 g dGMP 1 mg dUMP 50 g dUMP 1 mg TMP 50 g TMP 50 g Casein 1 mg
171 ⫾ 45 309 ⫾ 58 315 ⫾ 42 254 ⫾ 64 773 ⫾ 293 805 ⫾ 152 1172 ⫾ 98 226 ⫾ 12 177 ⫾ 54 345 ⫾ 32 586 ⫾ 55 141 ⫾ 34 472 ⫾ 81 245 ⫾ 18 222 ⫾ 32 760 ⫾ 68 871 ⫾ 97 128 ⫾ 52
0 0 0 0 299 ⫾ 21 598 ⫾ 64 653 ⫾ 32 0 0 153 ⫾ 55 212 ⫾ 33 92 ⫾ 34 161 ⫾ 12 0 0 349 ⫾ 23 289 ⫾ 41 0
INF-␥ (pg/mL) 109 ⫾ 31 88 ⫾ 40 0 0 340 ⫾ 73 438 ⫾ 31 348 ⫾ 21 1242 ⫾ 455 320 ⫾ 67 153 ⫾ 22 0 190 ⫾ 62 56 ⫾ 2 240 ⫾ 54 69 ⫾ 15 0 0 200 ⫾ 121
dAMP, deoxyadenosine monophosphate; dCMP, deoxycytidine monophosphate; dGMP, deoxyguanosine monophosphate; dUMP, deoxyuridine monophosphate; IFN-␥, interferon-␥; IL-10, interleukin-10; TMP, thymosine monophosphate; TNF-␣, tumor necrosis factor-␣ * Values are mean ⫾ standard error of the mean.
DNA/RNA and mononucleotide effects on PBMC cytokine secretion Healthy (Table 1) unstimulated (control) and casein-Na⫹ (control) stimulated PBMCs produced TNF-␣ and IFN-␥ in comparable amounts. Although these did not act like growth inducers, all nucleotide derivatives tested affected human PBMC cytokine secretion when compared with control and casein-Na⫹–stimulated cultures. All nucleotide derivatives (P ⬍ 0.05), but especially RNA and TMP, supported PBMC TNF-␣ secretion. In addition, RNA, dCMP, dGMP, and TMP induced IL-10 secretion, a cytokine that was not secreted by control PBMCs or those stimulated by DNA, dAMP, or dUMP. Only RNA and dAMP (particularly 50 g/mL) significantly (P ⬍ 0.05) triggered IFN-␥ secretion above control levels. DNA/RNA and mononucleotide effects on influenza virus antigen-induced PBMC cytokine secretion Influenza (Table 2) virus antigen (0.33 g/mL) induced no significant mean PBMC cytokine secretion above levels of control and casein-stimulated PBMCs. Some individual virus antigen-stimulated PBMCs showed significant differences in TNF-␣ secretion from control PBMCs, but this significance disappeared after calculating mean PBMC responses. RNA and dAMP cocultured with virus antigen significantly (P ⬍ 0.05) increased PBMC secretions of
IFN-␥, IL-10, and TNF-␣. Interestingly, compared with control, DNA increased (P ⬍ 0.05) virus antigen-induced cell secretion of IFN-␥ only, whereas dUMP significantly increased secretion of IL-10 only. IL-10 was not secreted by control cultures or PBMCs stimulated by virus antigen, dAMP, dUMP, or DNA (with or without virus antigen). Nucleotide dGMP completely inhibited virus-triggered cell secretion of IFN-␥ (P ⬍ 0.05), whereas TMP did not change the virus-induced secretion pattern of the measured cytokines compared with secretion of TMP alone and virus antigen alone.
Discussion In this study we found that DNA from fish soft roe, RNA from bakers yeast, and mononucleotides exerts direct action on healthy human immune cells in vitro. Using influenza virus antigen as a specific antigen, DNA, RNA, dAMP, dCMP, and dUMP increased antigen-induced PBMC proliferation responses. This corresponded with an increase in PBMC secretion of selected cytokines. RNA and dAMP acted as general cytokine inducers by increasing secretions of IFN-␥, TNF-␣, and IL-10, whereas DNA increased PBMC secretion of IFN-␥ only and dUMP stimulated secretion of IL-10 only. IL-10 was not secreted by
Table 2 DNA, RNA, and mononucleotides, in culture with Infl (0.33, g/mL), modulate secretion of TNF-␣, INF-␥, and IL-10 by peripheral blood mononuclear cells from 10 healthy subjects* Cultures
TNF-␣ (pg/mL)
IL-10 (pg/mL)
INF-␥ (pg/mL)
Control Casein Infl DNA 50 g ⫹ Infl DNA 1 mg ⫹ Infl DNA 5 mg ⫹ Infl RNA 50 g ⫹ Infl RNA 1 mg ⫹ Infl RNA 5 mg ⫹ Infl dAMP 50 g ⫹ Infl dAMP 1 mg ⫹ Infl dCMP 50 g ⫹ Infl dCMP 1 mg ⫹ Infl dGMP 50 g ⫹ Infl dGMP 1 mg ⫹ Infl dUMP 50 g ⫹ Infl dUMP 1 mg ⫹ Infl TMP 50 g ⫹ Infl TMP 1 mg ⫹ Infl
171 ⫾ 45 128 ⫾ 52 358 ⫾ 171 440 ⫾ 220 304 ⫾ 156 291 ⫾ 68 846 ⫾ 245 1038 ⫾ 420 1493 ⫾ 376 393 ⫾ 125 320 ⫾ 56 430 ⫾ 256 985 ⫾ 199 227 ⫾ 79 412 ⫾ 165 318 ⫾ 155 397 ⫾ 267 675 ⫾ 398 1122 ⫾ 412
0
109 ⫾ 31 200 ⫾ 121 159 ⫾ 56 309 ⫾ 78 412 ⫾ 123 298 ⫾ 145 593 ⫾ 245 553 ⫾ 156 839 ⫾ 298 592 ⫾ 222 1534 ⫾ 520 231 ⫾ 134 226 ⫾ 24 0 0 172 ⫾ 76 178 ⫾ 45 173 ⫾ 76 235 ⫾ 34
0 0 0 0 493 ⫾ 38 658 ⫾ 254 664 ⫾ 159 130 ⫾ 45 180 ⫾ 67 142 ⫾ 98 230 ⫾ 124 119 ⫾ 34 146 ⫾ 54 227 ⫾ 103 165 ⫾ 76 231 ⫾ 34 310 ⫾ 68
dAMP, deoxyadenosine monophosphate; dCMP, deoxycytidine monophosphate; dGMP, deoxyguanosine monophosphate; dUMP, deoxyuridine monophosphate; IFN-␥, interferon-␥; IL-10, interleukin-10; TMP, thymosine monophosphate; TNF-␣, tumor necrosis factor-␣ * Values are mean ⫾ standard error of the mean of triplicate measurements.
E. Holden et al. / Nutrition 21 (2005) 1003–1009
relevant control cultures, by dAMP- or dUMP-stimulated PBMCs, or by any of the cultures in which DNA was added with or without virus antigen. Nucleotides dGMP and TMP inhibited influenza virus-induced PBMC growth and dGMP completely blocked virus-induced IFN-␥ secretion. TMP did not change cytokine secretion in any culture. Our results indicated that coculturing virus antigen and nucleotides probably influence and activate distinct cell fractions in PBMCs and involve antigen-presenting cells and T cells. It is tempting to speculate that specific nucleotides may act as immunomodulators or costimulatory molecules by exerting their effect, perhaps mostly, on specific antigen-activated cells among PBMCs. Jyonouchi et al. [18] reported that yeast RNA enhances immunoglobulin (Ig) M and IgG production to T-cell– dependent stimuli but not to T-cell–independent stimuli. Decomposing RNA to pyrimidine bases decreased the enhancing action of RNA on Ig production. It has been proposed that polynucleotides modulate the process of antigenand allergen-mediated Th-cell activation, thus enhancing IFN-␥ secretion [12–14,19,20] In our investigation, the various DNA/RNA/nucleotide derivatives showed similar and different effects on immune cell growth and cytokine secretion, a behavior that also was observed when investigating nucleotide effects on intestinal cell lines [21]. This indicates that configuration and type of nucleotide bases influence the resulting effects. Immunostimulatory and “non-immunostimulatory” CpG synthetic oligonucleotides showing adjuvant effects were described by McCluskie et al. [22]. Further, prepriming and antigen covaccination with immunostimulatory DNA sequences promoted a Th1 response to antigen [23]. Therefore, the supporting ability of nucleotide derivatives on growth and cytokine secretion in our virus-stimulated immune cell cultures raises the possibility that specific sequences of DNA/RNA or specific nucleotides may act as potent Th1-directing adjuvants and converters of Th2 immune responses into Th1 responses. This has to be investigated further by stimulating human allergic PBMCs and different PBMC cell fractions with specific allergen and DNA/RNA/nucleotides and assessing cell proliferation and cytokine profiles. Further, influenza virus antigen-induced PBMC responses (Th1 like) should be compared with PBMC responses after measles virus infection (Th2 like). Thus, nucleotide sequences with optimal adjuvant activity could be detected. In our investigations, RNA from bakers yeast and dAMP acted as a general inducer of TNF-␣, IFN-␥, and IL-10, whereas DNA from fish soft roe seemed to be linked to regulation of IFN-␥ secretion in particular. The RNA effects observed resemble the wide cytokine-inducing ability of CpG-DNA described by Magnusson et al. [24]. Nucleotides dGMP and TMP had no apparent growth effect, but TMP supported high TNF-␣ secretion and dGMP cocultured with
1007
virus antigen inhibited IFN-␥ secretion. Several researchers have reported that immunostimulatory DNA suppresses IgE synthesis by promoting IgG, IFN-␥, IFN-␣, IFN-, IL-12, and IL-18 but also TNF-␣ secretion [25–27]. TNF-␣ has an unequivocal beneficial function in activating host defences because it can mediate resistance to infectious diseases by controlling intracellular pathogen multiplication and limiting the duration and extent of inflammatory processes [28]. However, overproduction of TNF-␣ can result in tissue damage and is followed by the emergence of IL-10. In septic shock, IL-10 decreases TNF-␣ levels [29]. This probably makes DNA from fish soft roe a better candidate for a vaccine adjuvant than RNA from bakers yeast or dAMP to avoid the generally high RNA cytokine-inducing ability. In any case, it should be possible to identify DNA/RNA/nucleotide sequences that activate beneficial cytokines differently to avoid secretion of “harmful” cytokines. The mechanism of action of immunostimulatory DNA is unknown. Nucleotide receptors (which respond to purines and pyrimidines) have a very widespread tissue distribution [20,22–32]. Natural nucleosides permeate mammalian cells by means of several carrier-mediated pathways [33] and soluble oligonucleotides, regardless of stimulating potential, bind to a B-cell surface receptor [34]. Recent information about the Toll-like receptor (TLR) family [35] has shown that TLR-9 [36] recognizes immunostimulatory CpG motifs, whereas TLR-3 recognizes viral double-stranded RNA [37]. Single-stranded RNA has been identified as the natural ligand for mouse TLR-7 and human TLR-8 [38,39]. Gelman et al. [40] found that stimulation with CpG-containing DNA increased the survival of a purified population of activated CD4⫹ T cells in vitro by increasing the viable cell number without changing proliferation. Emerging data have indicated that targeting TLR-3, TLR-4, and TLR-9 pathways can enhance responses to certain viral infections [41]. Most tissues express at least one TLR, whereas spleen and peripheral blood leukocytes and especially phagocytes express a great variety of TLRs. Engagement of TLR induces activation of nuclear factor-B and microtubule-associated protein kinase signaling pathways and cytokine production. However, striking differences have been reported in gene expression profile activation of TLRs by their specific ligands that produce differences in cytokine and chemokine expressions [42]. This may explain the qualitative and quantitative differences observed when measuring growth and cytokine secretion by the different DNA/RNA/nucleotide derivatives in our investigation. Although this has not been demonstrated, nucleotides and nucleotide fragments (depending on structure, concentration, and affinity) may act as ligands or costimulatory molecules for some or restricted TLRs. DNA from fish soft roe and RNA from bakers yeast and deoxy mononucleotides possess immunomodulatory properties that might be used in vaccine adjuvancy and in allergy protection.
1008
E. Holden et al. / Nutrition 21 (2005) 1003–1009
Conclusion The nucleotides used in this study did not, by themselves, induce any proliferation of PBMCs derived from healthy individuals. The nucleotides affected PBMC cytokine secretion. Cultured with a specific antigen, RNA, DNA, dAMP, dCMP, and dUMP increased antigen-induced PBMC proliferation responses and PBMC secretion of selected cytokines. DNA increased specific antigen-induced PBMC secretion of IFN-␥ only. Nucleotides dGMP and TMP inhibited antigen-induced PBMC growth and dGMP completely inhibited antigen-induced PBMC IFN-␥ secretion. TMP did not change antigen-induced PBMC secretion of the measured cytokines.
Acknowledgments The influenza virus antigen was kindly supported by Karl Brokstad, D.Phil., at Broegelmann Research Laboratory, University of Bergen.
References [1] Ardawi MS, Newsholme EA. Glutamine metabolism in lymphocytes of the rat. Biochem J 1983;212:835– 42. [2] Newsholme P, Gordon S, Newsholme EA. Rates of utilisation and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages. Biochem J 1987;242:631– 6. [3] Carver JD. Dietary nucleotides: cellular immune, intestinal and hepatic system effects. J Nutr 1994;124:144S– 8S. [4] Carver JD. Dietary nucleotide: effects on the immune and gastrointestinal systems. Acta Paediatr Suppl 1999;430:83– 8. [5] Jyonouchi H. Nucleotide actions on humoral immune responses. J Nutr 1994;124:138S– 43. [6] Van Buren CT, Kulkarni AD, Rudolph FB. The role of nucleotides in adult nutrition. J Nutr 1994;124:160S– 4. [7] Rudolph FB, Kulkarni AD, Fanslow WC, Pizzini RP, Kumar S, Van Buren CT. Role of RNA as a dietary source of pyrimidines and purines in immune function. Nutrition 1990;6:45–52. [8] Kulkarni AD, Fanslow WC, Rudolph FB, Van Buren CT. Effect of dietary nucleotides on response to bacterial infections. JPEN 1986; 10:169 –71. [9] Adjei AA, Takamine F, Yokoyama H, Chung SY, Sato L, Shinjo S, et al. Effect of intraperitoneally administered nucleoside-nucleotide on the recovery from methicillin-resistant Staphylococcus aureus strain 8985 N infection in mice. J Nutr Sci Vitaminol 1992;38:221–5. [10] Fanslow WC, Kulkarni AD, Van Buren CT, Rudolph FB. Effect of nucleotide restriction and supplementation on resistance to experimental murine candidiasis. JPEN 1988;12:49 –52. [11] Adjei AA, Jones JT, Enriquez FJ, Yamamoto S. Dietary nucleotides and nucleosides reduce Cryptosporidium parvum infection in dexamethasone immunosuppressed adult mice. Exp Parasitol 1999;92: 199 –208. [12] Jyonouchi H, Sun S, Winship T, Kuchan MJ. Dietary ribonucleotides modulate type 1 and type 2 T-helper cell responses against ovalbumin in young BALB/cJ mice. J Nutr 2001;131:1165–70.
[13] Sudo N, Aiba Y, Takaki A, Tanaka K, Yu X-N, Oyama N, et al. Dietary nucleic acids promote a shift in Th1/Th2 balance toward Th1-dominant immunity. Clin Exp Allergy 2000;30:979 – 87. [14] Jyonouchi H, Sun S, Aburi T, Winship T, Huchan MJ. Dietary nucleotides modulate antigen-specific type 1 and type 2 T-cell responses in young C57BL/6 mice. Nutrition 2000;16:442– 6. [15] Sakai M, Taniguchi K, Mamoto K, Ogawa H, Tabata M. Immunostimulant effects of nucleotide isolated from yeast RNA on carp, Cyprinus carpio L. J Fish Dis 2001;24:433– 8. [16] Burrells C, Williams PD, Southgate PJ, Wadsworth SL. Dietary nucleotides: a novel supplement in fish feeds. 2. Effects on vaccination, salt water transfer, growth rates and physiology of Atlantic salmon (Salmo salar L.). Aquaculture 2001;199:171– 84. [17] Pickering LK, Granoff DM, Erickson JR, Masor ML, Cordle CT, Schaller JP, et al. Modulation of immune system by human milk and infant formula containing nucleotides. Pediatrics 1998;101: 242–9. [18] Jyonouchi H, Zhang L, Tomita Y. Studies on immune modulating actions of RNA/nucleotides. RNA/nucleotides enhance in vitro immunoglobulin production by human peripheral blood mononuclear cells in response to T dependent stimuli. Pediatr Res 1993; 33:458 – 65. [19] Jyonouchi H, Sun S. The actions of polynucleotides on effector stage cloned murine T-helper cells differ in each subset and depend on antigen concentration. J Nutr 1997;127:411–7. [20] Magone MT, Chan C-C, Beck L, Whitcup SM, Raz E. Systemic or mucosal administration of immunostimulatory DNA inhibits early and late phases of murine allergic conjunctivitis. Eur J Immunol 2000;30:1841–50. [21] Holen E, Jonsson R. Dietary nucleotides and intestinal cell lines: I. Modulation of growth. Nutr Res 2004;24:197–207. [22] McCluskie MJ, Davis HL. Oral, intrarectal and intranasal immunisations using CpG and non-CpG oligodeoxynucleotides as adjuvants. Vaccine 2000;19:413–22. [23] Kobayashi H, Horner AA, Takabayashi K, Nguyen M-D, Huag E, Inman N, et al. Immunostimulatory DNA prepriming: a novel approach for prolonged Th1 biased immunity. Cell Immunol 1999;198: 69 –75. [24] Magnusson M, Magnusson S, Vallin H, Ronnblom L, Alm GV. Importance of CpG dinucleotides in activation of natural IFN-alfa producing cells by a lupus-related oligonucleotide. Scand J Immunol 2001;54:1–9. [25] Calder PC. Dietary nucleic acids and Th1/Th2 balance: a clue to cow’s milk allergy. Clin Exp Allergy 2000;30:908 –10. [26] Lipford GB, Sparwasser T, Bauer M, Zimmermann S, Koch ES, Heeg K, et al. Immunostimulatory DNA: sequence dependent production of potentially harmful or useful cytokines. Eur J Immunol 1997;27: 3420 – 6. [27] Saeij JPJ, Stet RJM, deVries BJ, VanMuisWinkel WB, Wiegertjes GF. Molecular and functional characterisation of carp TNF: a link between TNF polymorphism and trypan tolerance? Dev Comp Immunol 2003;27:29 – 41. [28] Derouich-Guergour D, Brenier-Pinchart MP, Ambroise-Thomas P, Pelloux H. Tumor necrosis factor alpha receptors: role in the physiopathology of protozoan parasite infections. Int J Parasitol 2001;31: 763–9. [29] North RA, Barnard EA. Nucleotide receptors. Curr Opin Neurobipol 1997;7:346 –57. [30] Do EL, Kontani JG, Lowry SF. Nutritional modulation of immunity and the inflammatory response. Nutrition 1998;14:545–50. [31] Gorodeski GI, Hopfer U, De Santis BJ, Eckert RL, Rorke EA, Utian WH. Biphasic regulation of paracellular permeability in human cervical cells by two distinct nucleotide receptors. Am J Physiol 1995; 268:1215–26. [32] Marriott I, Inscho EW, Bost KL. Extracellular uridine nucleotides
E. Holden et al. / Nutrition 21 (2005) 1003–1009
[33]
[34]
[35] [36] [37]
initiate cytokine production by murine dendritic cells. Cell Immunol 1999;195:147–56. Balimane PV, Sinko PJ. Involvement of multiple transporters in the oral absorption of nucleosides analogues. Adv Drug Deliv 1999;39: 183–209. Liang H, Reich CF, Pisetsky DS, Lipsky PE. The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides. J Immunol 2000;165:1438 – 45. Jefferies C, O’Neill LAJ. Signal transduction pathway activated by Toll-like receptors. Mod Aspects Immunobiol 2002;2:169 –75. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like receptor recognises bacterial DNA. Nature 2000;408:740 –5. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappa by Toll-like receptor 3. Nature 2001;413:732– 8.
1009
[38] Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004;303:1529 –31. [39] Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 2004;303:1526 –9. [40] Gelman AE, Zhang J, Choi Y, Turka LA. Toll-like receptor ligands directly promote activated CD4⫹ T cell survival. J Immunol 2004; 172:6065–73. [41] Ulevitch RJ. Therapeutics targeting the innate immune system. Nat Rev Immunol 2004;4:512–20. [42] Hirschfeld M, Weis JJ, Toshchakov V, Salowski CA, Cody MJ, Ward DC, et al. Signalling by toll-like receptor 2 and 4 agonists’ results in differential gene expression in murine macrophages. Inf Immun 2001; 69:1477– 82.
Applied nutritional investigation
Dietary nucleotides and human immune cells. II. Modulation of PBMC growth and cytokine secretion Elisabeth Holen, Ph.D.*, Oddvin A. Bjørge, M.D., and Roland Jonsson, Ph.D. Broegelmann Research Laboratory, University of Bergen, Bergen, Norway Manuscript received October 21, 2004; accepted March 24, 2005.
Abstract
Objective: The immune system is dependent on purines and pyrimidines as building blocks for DNA and RNA synthesis to enable rapid cell proliferation and protein synthesis. Emerging evidence suggests that dietary nucleotides optimize immune function. We investigated whether growth and function of human immune cells were affected by an exogenous source of nucleotides during specific antigen challenge. Methods: Peripheral blood mononuclear cells from healthy individuals (n ⫽ 10) were stimulated with influenza virus antigen and DNA-Na⫹ from fish soft roe, RNA from bakers yeast (Saccharomyces cerevisiae), 2=deoxyadenosine 5=-monophosphate sodium, 2=deoxycytidine 5=-monophosphate sodium, 2=deoxyguanosine 5=-monophosphate sodium, or 2=deoxyuridine 5=-monophosphate disodium. Growth effects were ascertained by measuring the amount of tritium-labeled Thymidine 5=-monophosphate sodium incorporated into cell DNA. Cell function was measured by detection of interferon-␥ (IFN-␥), tumor necrosis factor-␣, and interleukin-10 production. Results: Specific nucleotide derivatives alone did not affect the growth of healthy peripheral blood mononuclear cells. However, the nucleotide derivatives influenced immune cell growth and cytokine secretion when cocultured with specific antigen. DNA, RNA, deoxyadenosine monophosphate, deoxycytidine monophosphate, and deoxyuridine monophosphate increased influenza virus antigeninduced immune cell proliferation. In contrast, deoxyadenosine monophosphate and thymosine monophosphate inhibited the antigen-induced growth response. RNA and deoxyadenosine monophosphate cocultured with virus antigen significantly increased peripheral blood mononuclear cell secretion of IFN-␥, interleukin-10, and tumor necrosis factor-␣. DNA increased virus antigeninduced immune cell secretion of IFN-␥ only, whereas deoxyuridine monophosphate significantly increased secretion of interleukin-10 only. Deoxyguanosine monophosphate completely inhibited virus-triggered IFN-␥ secretion, whereas thymosine monophosphate did not change the secretion pattern of measured cytokines. Conclusion: Nucleotide derivatives affect growth and function of specific virus antigen-stimulated human immune cells in vitro. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Peripheral blood mononuclear cells; DNA; RNA; Nucleotides; Influenza virus antigen
Introduction The immune system is dependent on adequate nutrition for different vital processes such as energy production, protein synthesis, proliferation, and other specific metabolic
This study was supported by Bjørge Biomarin AS. * Corresponding author. Tel.: ⫹47-55-905-134; fax: ⫹47-55-905-299. E-mail address: [email protected] (E. Holen). 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2005.03.003
pathways. Immune cells require several nutrients to increase their growth rate and function [1,2]. Dietary nucleic acids and nucleotides influence optimal growth and cell function [3,4]. In rodents, nucleotide-free diets decrease cellular and humoral immune responses [5–7] and resistance to bacterial and fungal pathogens [8 –11]. Further, dietary nucleotides can modulate type 1 and type 2 T-helper (Th) cell responses by promoting a shift in the Th1/Th2 balance toward Th1dominant immunity [12–14]. In fish, dietary nucleotides seem to enhance non-specific immune responses [15] and
1004
E. Holden et al. / Nutrition 21 (2005) 1003–1009
vaccine efficacy [16]. In humans, dietary nucleotides increase immune responses to Haemophilia influenzae virus b and diphtheria [17], and polynucleotides significantly increase in vitro antibody production in response to T-cell– dependent antigen [9]. Nucleotides, in addition to being involved in well-known energy-dependent processes, exhibit functions as pluripotent signaling molecules that elicit direct effects on cell functions by purine and pyrimidine receptors but probably also other receptors to induce gene expression. Because dietary nucleotides may exert a wide range of effects depending on nucleotide concentration, structure, and possible receptor affinity, reflected by their target cells, it is necessary to evaluate the effect of various nucleotides on different cell types. In this study we investigated DNA isolated from fish soft roe, RNA from bakers yeast, 2=deoxyadenosine 5=-monophosphate sodium (dAMP-Na⫹), 2=deoxycytidine 5=-monophosphate sodium (dCMP-Na⫹), 2=deoxyguanosine 5=monophosphate sodium (dGMP-Na⫹), 2=deoxyuridine 5=monophosphate disodium (dUMP-Na⫹), and Thymidine 5=monophosphate sodium (TMP-Na⫹) to determine their capacity to stimulate immune cell growth and function and used influenza virus antigen as an immunostimulant in vitro.
Materials and methods DNA and RNA derivatives Sodium salt of DNA from fish soft roe (Bjørge Biomarin AS). Nucleotides dUMP-Na⫹ (D3876), dGMP-Na⫹ (D9500), dCMP-Na⫹ (D7625), dAMP-Na⫹ (D6250), TMP-Na⫹ (T7004) and RNA type III from bakers yeast (R7125) were purchased from Sigma (St. Louis, MO, USA). TMP in this study was used because of unpublished results that indicated effects on growth and immune function in rats fed a TMPsupplemented diet. Initially, all nucleotide derivatives were titrated to find optimal concentrations for cell culture analysis and were estimated from trials that included virus antigen because no proliferation responses were observed with nucleotides and peripheral blood mononuclear cells (PBMCs) alone. To visualize different concentrations of nucleotides, we used 50 g/mL, 1 mg/mL, and 5 mg/mL as suitable concentrations of DNA and RNA for cell culture analysis. Higher concentrations (up to 20 mg/mL) did not essentially change cell growth or function. Nucleotides were used in final concentrations of 50 g/mL and 1 mg/mL.
proliferation response in most PBMCs tested. The virus antigen solution of 1.3 g/mL was the concentration at which most PBMCs showed a significant lower response to virus antigen, compared with the 0.33-g/mL dose, probably due to an earlier onset of cell damage and nonresponsiveness to nucleotide addition. Cells PBMCs from 10 healthy volunteers were isolated from the interface after Ficoll-Paque gradient centrifugation (Pharmacia LKB, Uppsala, Sweden), washed, and resuspended in RMPI-1640 medium supplemented with 10% heat inactivated foetal calf serum and 1% of a 200-mM L-glutamine solution (Gibco BRL, Paisley, UK). Proliferation assay PBMCs (⬎1 ⫻ 105 cells/well) were cultured in 24-well culture clusters (Costar, Cambridge, MA, USA) with or without antigen/DNA/nucleotides and RNA in the concentrations described to a final volume of 2 mL. All reagents were added at initiation of cultures. Five days after initiating cultures, each culture was mixed with a pipette. From each well, 200 L in triplicate was seeded into 96-well cell culture clusters (Costar) and pulsed overnight by adding 20 L of 0.05 mCi/mL [methyl-3H]-thymidine (Amersham, Copenhagen, Denmark). Cultures were harvested into filter mats by using a semiautomatic cell harvester (Skatron, Lier, Norway). Harvested filters and 3 mL of an Ultima Gold liquid scintillation cocktail (Packard Instruments, Downers Grove, IL, USA) were counted in Skatron scintillation vials by a liquid scintillation counter (LKB Wallac 1217, Rackbeta, Bromma, Sweden). Proliferation responses Results are presented as determinations per minute (dpm) means of 10 individual PBMCs. Each PBMC was measured in triplicate as the dpm mean ⫾ standard error of the mean. Unstimulated PBMCs and PBMCs stimulated by an unrelated protein, casein-Na⫹, were included as controls. PBMC supernatants The 24-well PBMC cultures were harvested on day 6 and cells were removed by centrifugation. Cell supernatants were stored at ⫺20°C for cytokine profile analysis. Cytokine analysis
Antigens Influenza virus antigen (A/Beijing/262/95, HxH12W) at final concentrations of 0.33 and 1.3 g/mL of culture was used as the stimulating antigen. Virus antigen diluted to 0.33 g/mL was the concentration that produced the highest
Estimations of interferon-␥ (IFN-␥; DY285, detection limit 15.6 pg/mL), tumor necrosis factor-␣ (TNF-␣; DY210, detection limit 15.6 pg/mL), and interleukin-10 (IL-10; DY217, detection limit 46.9 pg/mL) in culture supernatants of human PBMCs were performed with commer-
E. Holden et al. / Nutrition 21 (2005) 1003–1009
Fig. 1. DNA-Na⫹ from fish soft roe (50 g/mL) and RNA from bakers yeast (50 g/mL or 1 mg/mL) significantly increases Inf-induced immune cell growth. Inf (0.33 g/mL) induced significant proliferation of peripheral blood mononuclear cells compared with control casein (P ⫽ 0.0367), Triplicate measurements of peripheral blood mononuclear cells from 10 healthy subjects were investigated. Results are presented as mean dpm ⫾ standard error of the mean. Differences are significant at P ⬍ 0.05. dpm, determinations per minute; Inf, influenza virus antigen.
cial enzyme-linked immunosorbent assay kits (R&D, London, UK) according to the manufacturer’s instructions. The manufacturer provided the specificity and sensitivity of the kits. Statistics The computer program GrapPad Prism 3.0a (Graph Pad Software, Inc., San Diego, CA, USA) was used for statistical analysis of significant differences. Two-way analysis of variance and t test (to compare each mean with the control mean) were used. P ⬍ 0.05 was considered statistically significant.
1005
Fig. 2. Nucleotides dAMP, dCMP (50 g/mL, 1 mg/mL), and dUMP (50 g/mL) significantly increase Inf-induced immune cell growth. Triplicate measurements of peripheral blood mononuclear cells from 10 healthy subjects were investigated. Growth responses are presented as mean dpm ⫾ standard error of the mean. Differences are significant at P ⬍ 0.05. dAMP, deoxyadenosine monophosphate; dCMP, deoxycytidine monophosphate; dpm, ??; dUMP, deoxyuridine monophosphate; Inf, influenza virus antigen.
in particular, but also 1 mg/mL (except 1 mg/mL of dUMP and DNA), significantly triggered virus-induced PBMC growth. The highest proliferation responses, compared to virus antigen alone (1897 mean dpm) were observed in PBMC cultures in which RNA, dAMP, and dCMP were added with virus antigen (3970-, 4326-, 4086- mean dpm respectively, Figs. 1 and 2). In contrast, dGMP and TMP (at all concentrations tested) showed no significant effect on influenza virus-antigen induced growth responses (Fig. 3).
Results Growth responses DNA-Na⫹ from fish soft roe, RNA from bakers yeast, or mononucleotides did not trigger growth of PBMCs from healthy subjects (n ⫽ 10, P ⬎ 0.05; Fig. 1). Influenza virus antigen (0.33 g/mL) induced significant immune cell proliferation compared with the control casein (P ⫽ 0.0367). In contrast, 1.3 g/mL of virus antigen produced a three-fold lower mean response (Fig. 1) and was not significantly different from the mean of the control group. Interestingly, when cultured with virus antigen, DNA-Na⫹, and RNA (Fig. 1), dAMP, dCMP, and dUMP (Fig. 2) significantly (P ⬍ 0.05) increased virus antigen-induced PBMC proliferation responses. Nucleotide concentrations of 50 g/mL
Fig. 3. Nucleotides dGMP and TMP, when cocultured with Inf, inhibit virus-induced growth of peripheral blood mononuclear cells. Responses are presented as dpm mean ⫾ standard error of the mean (n ⫽ 10, P ⬍ 0.05). dGMP, deoxyguanosine monophosphate; dpm, ??; Inf, influenza virus antigen; TMP, thymosine monophosphate.
1006
E. Holden et al. / Nutrition 21 (2005) 1003–1009
Table 1 DNA-, RNA-, and mononucleotide-induced secretion of TNF-␣, IL-10, and INF-␥ by peripheral blood mononuclear cells from 10 healthy subjects* Culture
TNF-␣ (pg/mL)
IL-10 (pg/mL)
Control DNA 50 g DNA 1 mg DNA 5 mg RNA 50 g RNA 1 mg RNA 5 mg dAMP 50 g dAMP 1 mg dCMP 50 g dCMP 1 mg dGMP 50 g dGMP 1 mg dUMP 50 g dUMP 1 mg TMP 50 g TMP 50 g Casein 1 mg
171 ⫾ 45 309 ⫾ 58 315 ⫾ 42 254 ⫾ 64 773 ⫾ 293 805 ⫾ 152 1172 ⫾ 98 226 ⫾ 12 177 ⫾ 54 345 ⫾ 32 586 ⫾ 55 141 ⫾ 34 472 ⫾ 81 245 ⫾ 18 222 ⫾ 32 760 ⫾ 68 871 ⫾ 97 128 ⫾ 52
0 0 0 0 299 ⫾ 21 598 ⫾ 64 653 ⫾ 32 0 0 153 ⫾ 55 212 ⫾ 33 92 ⫾ 34 161 ⫾ 12 0 0 349 ⫾ 23 289 ⫾ 41 0
INF-␥ (pg/mL) 109 ⫾ 31 88 ⫾ 40 0 0 340 ⫾ 73 438 ⫾ 31 348 ⫾ 21 1242 ⫾ 455 320 ⫾ 67 153 ⫾ 22 0 190 ⫾ 62 56 ⫾ 2 240 ⫾ 54 69 ⫾ 15 0 0 200 ⫾ 121
dAMP, deoxyadenosine monophosphate; dCMP, deoxycytidine monophosphate; dGMP, deoxyguanosine monophosphate; dUMP, deoxyuridine monophosphate; IFN-␥, interferon-␥; IL-10, interleukin-10; TMP, thymosine monophosphate; TNF-␣, tumor necrosis factor-␣ * Values are mean ⫾ standard error of the mean.
DNA/RNA and mononucleotide effects on PBMC cytokine secretion Healthy (Table 1) unstimulated (control) and casein-Na⫹ (control) stimulated PBMCs produced TNF-␣ and IFN-␥ in comparable amounts. Although these did not act like growth inducers, all nucleotide derivatives tested affected human PBMC cytokine secretion when compared with control and casein-Na⫹–stimulated cultures. All nucleotide derivatives (P ⬍ 0.05), but especially RNA and TMP, supported PBMC TNF-␣ secretion. In addition, RNA, dCMP, dGMP, and TMP induced IL-10 secretion, a cytokine that was not secreted by control PBMCs or those stimulated by DNA, dAMP, or dUMP. Only RNA and dAMP (particularly 50 g/mL) significantly (P ⬍ 0.05) triggered IFN-␥ secretion above control levels. DNA/RNA and mononucleotide effects on influenza virus antigen-induced PBMC cytokine secretion Influenza (Table 2) virus antigen (0.33 g/mL) induced no significant mean PBMC cytokine secretion above levels of control and casein-stimulated PBMCs. Some individual virus antigen-stimulated PBMCs showed significant differences in TNF-␣ secretion from control PBMCs, but this significance disappeared after calculating mean PBMC responses. RNA and dAMP cocultured with virus antigen significantly (P ⬍ 0.05) increased PBMC secretions of
IFN-␥, IL-10, and TNF-␣. Interestingly, compared with control, DNA increased (P ⬍ 0.05) virus antigen-induced cell secretion of IFN-␥ only, whereas dUMP significantly increased secretion of IL-10 only. IL-10 was not secreted by control cultures or PBMCs stimulated by virus antigen, dAMP, dUMP, or DNA (with or without virus antigen). Nucleotide dGMP completely inhibited virus-triggered cell secretion of IFN-␥ (P ⬍ 0.05), whereas TMP did not change the virus-induced secretion pattern of the measured cytokines compared with secretion of TMP alone and virus antigen alone.
Discussion In this study we found that DNA from fish soft roe, RNA from bakers yeast, and mononucleotides exerts direct action on healthy human immune cells in vitro. Using influenza virus antigen as a specific antigen, DNA, RNA, dAMP, dCMP, and dUMP increased antigen-induced PBMC proliferation responses. This corresponded with an increase in PBMC secretion of selected cytokines. RNA and dAMP acted as general cytokine inducers by increasing secretions of IFN-␥, TNF-␣, and IL-10, whereas DNA increased PBMC secretion of IFN-␥ only and dUMP stimulated secretion of IL-10 only. IL-10 was not secreted by
Table 2 DNA, RNA, and mononucleotides, in culture with Infl (0.33, g/mL), modulate secretion of TNF-␣, INF-␥, and IL-10 by peripheral blood mononuclear cells from 10 healthy subjects* Cultures
TNF-␣ (pg/mL)
IL-10 (pg/mL)
INF-␥ (pg/mL)
Control Casein Infl DNA 50 g ⫹ Infl DNA 1 mg ⫹ Infl DNA 5 mg ⫹ Infl RNA 50 g ⫹ Infl RNA 1 mg ⫹ Infl RNA 5 mg ⫹ Infl dAMP 50 g ⫹ Infl dAMP 1 mg ⫹ Infl dCMP 50 g ⫹ Infl dCMP 1 mg ⫹ Infl dGMP 50 g ⫹ Infl dGMP 1 mg ⫹ Infl dUMP 50 g ⫹ Infl dUMP 1 mg ⫹ Infl TMP 50 g ⫹ Infl TMP 1 mg ⫹ Infl
171 ⫾ 45 128 ⫾ 52 358 ⫾ 171 440 ⫾ 220 304 ⫾ 156 291 ⫾ 68 846 ⫾ 245 1038 ⫾ 420 1493 ⫾ 376 393 ⫾ 125 320 ⫾ 56 430 ⫾ 256 985 ⫾ 199 227 ⫾ 79 412 ⫾ 165 318 ⫾ 155 397 ⫾ 267 675 ⫾ 398 1122 ⫾ 412
0
109 ⫾ 31 200 ⫾ 121 159 ⫾ 56 309 ⫾ 78 412 ⫾ 123 298 ⫾ 145 593 ⫾ 245 553 ⫾ 156 839 ⫾ 298 592 ⫾ 222 1534 ⫾ 520 231 ⫾ 134 226 ⫾ 24 0 0 172 ⫾ 76 178 ⫾ 45 173 ⫾ 76 235 ⫾ 34
0 0 0 0 493 ⫾ 38 658 ⫾ 254 664 ⫾ 159 130 ⫾ 45 180 ⫾ 67 142 ⫾ 98 230 ⫾ 124 119 ⫾ 34 146 ⫾ 54 227 ⫾ 103 165 ⫾ 76 231 ⫾ 34 310 ⫾ 68
dAMP, deoxyadenosine monophosphate; dCMP, deoxycytidine monophosphate; dGMP, deoxyguanosine monophosphate; dUMP, deoxyuridine monophosphate; IFN-␥, interferon-␥; IL-10, interleukin-10; TMP, thymosine monophosphate; TNF-␣, tumor necrosis factor-␣ * Values are mean ⫾ standard error of the mean of triplicate measurements.
E. Holden et al. / Nutrition 21 (2005) 1003–1009
relevant control cultures, by dAMP- or dUMP-stimulated PBMCs, or by any of the cultures in which DNA was added with or without virus antigen. Nucleotides dGMP and TMP inhibited influenza virus-induced PBMC growth and dGMP completely blocked virus-induced IFN-␥ secretion. TMP did not change cytokine secretion in any culture. Our results indicated that coculturing virus antigen and nucleotides probably influence and activate distinct cell fractions in PBMCs and involve antigen-presenting cells and T cells. It is tempting to speculate that specific nucleotides may act as immunomodulators or costimulatory molecules by exerting their effect, perhaps mostly, on specific antigen-activated cells among PBMCs. Jyonouchi et al. [18] reported that yeast RNA enhances immunoglobulin (Ig) M and IgG production to T-cell– dependent stimuli but not to T-cell–independent stimuli. Decomposing RNA to pyrimidine bases decreased the enhancing action of RNA on Ig production. It has been proposed that polynucleotides modulate the process of antigenand allergen-mediated Th-cell activation, thus enhancing IFN-␥ secretion [12–14,19,20] In our investigation, the various DNA/RNA/nucleotide derivatives showed similar and different effects on immune cell growth and cytokine secretion, a behavior that also was observed when investigating nucleotide effects on intestinal cell lines [21]. This indicates that configuration and type of nucleotide bases influence the resulting effects. Immunostimulatory and “non-immunostimulatory” CpG synthetic oligonucleotides showing adjuvant effects were described by McCluskie et al. [22]. Further, prepriming and antigen covaccination with immunostimulatory DNA sequences promoted a Th1 response to antigen [23]. Therefore, the supporting ability of nucleotide derivatives on growth and cytokine secretion in our virus-stimulated immune cell cultures raises the possibility that specific sequences of DNA/RNA or specific nucleotides may act as potent Th1-directing adjuvants and converters of Th2 immune responses into Th1 responses. This has to be investigated further by stimulating human allergic PBMCs and different PBMC cell fractions with specific allergen and DNA/RNA/nucleotides and assessing cell proliferation and cytokine profiles. Further, influenza virus antigen-induced PBMC responses (Th1 like) should be compared with PBMC responses after measles virus infection (Th2 like). Thus, nucleotide sequences with optimal adjuvant activity could be detected. In our investigations, RNA from bakers yeast and dAMP acted as a general inducer of TNF-␣, IFN-␥, and IL-10, whereas DNA from fish soft roe seemed to be linked to regulation of IFN-␥ secretion in particular. The RNA effects observed resemble the wide cytokine-inducing ability of CpG-DNA described by Magnusson et al. [24]. Nucleotides dGMP and TMP had no apparent growth effect, but TMP supported high TNF-␣ secretion and dGMP cocultured with
1007
virus antigen inhibited IFN-␥ secretion. Several researchers have reported that immunostimulatory DNA suppresses IgE synthesis by promoting IgG, IFN-␥, IFN-␣, IFN-, IL-12, and IL-18 but also TNF-␣ secretion [25–27]. TNF-␣ has an unequivocal beneficial function in activating host defences because it can mediate resistance to infectious diseases by controlling intracellular pathogen multiplication and limiting the duration and extent of inflammatory processes [28]. However, overproduction of TNF-␣ can result in tissue damage and is followed by the emergence of IL-10. In septic shock, IL-10 decreases TNF-␣ levels [29]. This probably makes DNA from fish soft roe a better candidate for a vaccine adjuvant than RNA from bakers yeast or dAMP to avoid the generally high RNA cytokine-inducing ability. In any case, it should be possible to identify DNA/RNA/nucleotide sequences that activate beneficial cytokines differently to avoid secretion of “harmful” cytokines. The mechanism of action of immunostimulatory DNA is unknown. Nucleotide receptors (which respond to purines and pyrimidines) have a very widespread tissue distribution [20,22–32]. Natural nucleosides permeate mammalian cells by means of several carrier-mediated pathways [33] and soluble oligonucleotides, regardless of stimulating potential, bind to a B-cell surface receptor [34]. Recent information about the Toll-like receptor (TLR) family [35] has shown that TLR-9 [36] recognizes immunostimulatory CpG motifs, whereas TLR-3 recognizes viral double-stranded RNA [37]. Single-stranded RNA has been identified as the natural ligand for mouse TLR-7 and human TLR-8 [38,39]. Gelman et al. [40] found that stimulation with CpG-containing DNA increased the survival of a purified population of activated CD4⫹ T cells in vitro by increasing the viable cell number without changing proliferation. Emerging data have indicated that targeting TLR-3, TLR-4, and TLR-9 pathways can enhance responses to certain viral infections [41]. Most tissues express at least one TLR, whereas spleen and peripheral blood leukocytes and especially phagocytes express a great variety of TLRs. Engagement of TLR induces activation of nuclear factor-B and microtubule-associated protein kinase signaling pathways and cytokine production. However, striking differences have been reported in gene expression profile activation of TLRs by their specific ligands that produce differences in cytokine and chemokine expressions [42]. This may explain the qualitative and quantitative differences observed when measuring growth and cytokine secretion by the different DNA/RNA/nucleotide derivatives in our investigation. Although this has not been demonstrated, nucleotides and nucleotide fragments (depending on structure, concentration, and affinity) may act as ligands or costimulatory molecules for some or restricted TLRs. DNA from fish soft roe and RNA from bakers yeast and deoxy mononucleotides possess immunomodulatory properties that might be used in vaccine adjuvancy and in allergy protection.
1008
E. Holden et al. / Nutrition 21 (2005) 1003–1009
Conclusion The nucleotides used in this study did not, by themselves, induce any proliferation of PBMCs derived from healthy individuals. The nucleotides affected PBMC cytokine secretion. Cultured with a specific antigen, RNA, DNA, dAMP, dCMP, and dUMP increased antigen-induced PBMC proliferation responses and PBMC secretion of selected cytokines. DNA increased specific antigen-induced PBMC secretion of IFN-␥ only. Nucleotides dGMP and TMP inhibited antigen-induced PBMC growth and dGMP completely inhibited antigen-induced PBMC IFN-␥ secretion. TMP did not change antigen-induced PBMC secretion of the measured cytokines.
Acknowledgments The influenza virus antigen was kindly supported by Karl Brokstad, D.Phil., at Broegelmann Research Laboratory, University of Bergen.
References [1] Ardawi MS, Newsholme EA. Glutamine metabolism in lymphocytes of the rat. Biochem J 1983;212:835– 42. [2] Newsholme P, Gordon S, Newsholme EA. Rates of utilisation and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages. Biochem J 1987;242:631– 6. [3] Carver JD. Dietary nucleotides: cellular immune, intestinal and hepatic system effects. J Nutr 1994;124:144S– 8S. [4] Carver JD. Dietary nucleotide: effects on the immune and gastrointestinal systems. Acta Paediatr Suppl 1999;430:83– 8. [5] Jyonouchi H. Nucleotide actions on humoral immune responses. J Nutr 1994;124:138S– 43. [6] Van Buren CT, Kulkarni AD, Rudolph FB. The role of nucleotides in adult nutrition. J Nutr 1994;124:160S– 4. [7] Rudolph FB, Kulkarni AD, Fanslow WC, Pizzini RP, Kumar S, Van Buren CT. Role of RNA as a dietary source of pyrimidines and purines in immune function. Nutrition 1990;6:45–52. [8] Kulkarni AD, Fanslow WC, Rudolph FB, Van Buren CT. Effect of dietary nucleotides on response to bacterial infections. JPEN 1986; 10:169 –71. [9] Adjei AA, Takamine F, Yokoyama H, Chung SY, Sato L, Shinjo S, et al. Effect of intraperitoneally administered nucleoside-nucleotide on the recovery from methicillin-resistant Staphylococcus aureus strain 8985 N infection in mice. J Nutr Sci Vitaminol 1992;38:221–5. [10] Fanslow WC, Kulkarni AD, Van Buren CT, Rudolph FB. Effect of nucleotide restriction and supplementation on resistance to experimental murine candidiasis. JPEN 1988;12:49 –52. [11] Adjei AA, Jones JT, Enriquez FJ, Yamamoto S. Dietary nucleotides and nucleosides reduce Cryptosporidium parvum infection in dexamethasone immunosuppressed adult mice. Exp Parasitol 1999;92: 199 –208. [12] Jyonouchi H, Sun S, Winship T, Kuchan MJ. Dietary ribonucleotides modulate type 1 and type 2 T-helper cell responses against ovalbumin in young BALB/cJ mice. J Nutr 2001;131:1165–70.
[13] Sudo N, Aiba Y, Takaki A, Tanaka K, Yu X-N, Oyama N, et al. Dietary nucleic acids promote a shift in Th1/Th2 balance toward Th1-dominant immunity. Clin Exp Allergy 2000;30:979 – 87. [14] Jyonouchi H, Sun S, Aburi T, Winship T, Huchan MJ. Dietary nucleotides modulate antigen-specific type 1 and type 2 T-cell responses in young C57BL/6 mice. Nutrition 2000;16:442– 6. [15] Sakai M, Taniguchi K, Mamoto K, Ogawa H, Tabata M. Immunostimulant effects of nucleotide isolated from yeast RNA on carp, Cyprinus carpio L. J Fish Dis 2001;24:433– 8. [16] Burrells C, Williams PD, Southgate PJ, Wadsworth SL. Dietary nucleotides: a novel supplement in fish feeds. 2. Effects on vaccination, salt water transfer, growth rates and physiology of Atlantic salmon (Salmo salar L.). Aquaculture 2001;199:171– 84. [17] Pickering LK, Granoff DM, Erickson JR, Masor ML, Cordle CT, Schaller JP, et al. Modulation of immune system by human milk and infant formula containing nucleotides. Pediatrics 1998;101: 242–9. [18] Jyonouchi H, Zhang L, Tomita Y. Studies on immune modulating actions of RNA/nucleotides. RNA/nucleotides enhance in vitro immunoglobulin production by human peripheral blood mononuclear cells in response to T dependent stimuli. Pediatr Res 1993; 33:458 – 65. [19] Jyonouchi H, Sun S. The actions of polynucleotides on effector stage cloned murine T-helper cells differ in each subset and depend on antigen concentration. J Nutr 1997;127:411–7. [20] Magone MT, Chan C-C, Beck L, Whitcup SM, Raz E. Systemic or mucosal administration of immunostimulatory DNA inhibits early and late phases of murine allergic conjunctivitis. Eur J Immunol 2000;30:1841–50. [21] Holen E, Jonsson R. Dietary nucleotides and intestinal cell lines: I. Modulation of growth. Nutr Res 2004;24:197–207. [22] McCluskie MJ, Davis HL. Oral, intrarectal and intranasal immunisations using CpG and non-CpG oligodeoxynucleotides as adjuvants. Vaccine 2000;19:413–22. [23] Kobayashi H, Horner AA, Takabayashi K, Nguyen M-D, Huag E, Inman N, et al. Immunostimulatory DNA prepriming: a novel approach for prolonged Th1 biased immunity. Cell Immunol 1999;198: 69 –75. [24] Magnusson M, Magnusson S, Vallin H, Ronnblom L, Alm GV. Importance of CpG dinucleotides in activation of natural IFN-alfa producing cells by a lupus-related oligonucleotide. Scand J Immunol 2001;54:1–9. [25] Calder PC. Dietary nucleic acids and Th1/Th2 balance: a clue to cow’s milk allergy. Clin Exp Allergy 2000;30:908 –10. [26] Lipford GB, Sparwasser T, Bauer M, Zimmermann S, Koch ES, Heeg K, et al. Immunostimulatory DNA: sequence dependent production of potentially harmful or useful cytokines. Eur J Immunol 1997;27: 3420 – 6. [27] Saeij JPJ, Stet RJM, deVries BJ, VanMuisWinkel WB, Wiegertjes GF. Molecular and functional characterisation of carp TNF: a link between TNF polymorphism and trypan tolerance? Dev Comp Immunol 2003;27:29 – 41. [28] Derouich-Guergour D, Brenier-Pinchart MP, Ambroise-Thomas P, Pelloux H. Tumor necrosis factor alpha receptors: role in the physiopathology of protozoan parasite infections. Int J Parasitol 2001;31: 763–9. [29] North RA, Barnard EA. Nucleotide receptors. Curr Opin Neurobipol 1997;7:346 –57. [30] Do EL, Kontani JG, Lowry SF. Nutritional modulation of immunity and the inflammatory response. Nutrition 1998;14:545–50. [31] Gorodeski GI, Hopfer U, De Santis BJ, Eckert RL, Rorke EA, Utian WH. Biphasic regulation of paracellular permeability in human cervical cells by two distinct nucleotide receptors. Am J Physiol 1995; 268:1215–26. [32] Marriott I, Inscho EW, Bost KL. Extracellular uridine nucleotides
E. Holden et al. / Nutrition 21 (2005) 1003–1009
[33]
[34]
[35] [36] [37]
initiate cytokine production by murine dendritic cells. Cell Immunol 1999;195:147–56. Balimane PV, Sinko PJ. Involvement of multiple transporters in the oral absorption of nucleosides analogues. Adv Drug Deliv 1999;39: 183–209. Liang H, Reich CF, Pisetsky DS, Lipsky PE. The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides. J Immunol 2000;165:1438 – 45. Jefferies C, O’Neill LAJ. Signal transduction pathway activated by Toll-like receptors. Mod Aspects Immunobiol 2002;2:169 –75. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like receptor recognises bacterial DNA. Nature 2000;408:740 –5. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappa by Toll-like receptor 3. Nature 2001;413:732– 8.
1009
[38] Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004;303:1529 –31. [39] Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 2004;303:1526 –9. [40] Gelman AE, Zhang J, Choi Y, Turka LA. Toll-like receptor ligands directly promote activated CD4⫹ T cell survival. J Immunol 2004; 172:6065–73. [41] Ulevitch RJ. Therapeutics targeting the innate immune system. Nat Rev Immunol 2004;4:512–20. [42] Hirschfeld M, Weis JJ, Toshchakov V, Salowski CA, Cody MJ, Ward DC, et al. Signalling by toll-like receptor 2 and 4 agonists’ results in differential gene expression in murine macrophages. Inf Immun 2001; 69:1477– 82.