Cellular Immunology 279 (2012) 30–41

Contents lists available at SciVerse ScienceDirect

Cellular Immunology

journal homepage: www.elsevier .com/ locate/yc imm

D-Limonene modulates T lymphocyte activity and viability

Courtney M. Lappas a,⇑, Nicholas T. Lappas b

a Department of Biology, Lebanon Valley College, Annville, PA 17003, United Statesb Department of Forensic Sciences, The George Washington University, Washington, DC 20007, United States

a r t i c l e i n f o

Article history:Received 3 December 2011Accepted 4 September 2012Available online 18 September 2012

Keywords:D-LimoneneImmuntoxicologyT lymphocytes

0008-8749/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.cellimm.2012.09.002

⇑ Corresponding author. Address: Department of Bio101 N. College Avenue, Annville, PA 17003, United St

E-mail address: lappas@lvc.edu (C.M. Lappas).

a b s t r a c t

D-Limonene, a cyclic terpene that is a major component of several plant essential oils, is used widely as anadditive in perfumes, soaps, foods and beverages, and has also been shown to possess chemopreventativeand chemotherapeutic activity. A limited number of studies have been conducted investigating the effectof D-limonene on immune system function. We show that D-limonene and its metabolites limonene-1-2-diol and perillic acid inhibit the production by CD3+CD4+ T cells of IFN-c, IL-2, TNF-a, IL-4 and IL-13, andthe production by CD3+CD8+ T cells of IFN-c, IL-2, and TNF-a. Additionally, the upregulation of CD25,CD69 and CD40L by activated T lymphocytes is modulated by D-limonene, limonene-1-2-diol and perillicacid treatment. Furthermore, high concentrations of D-limonene, limonene-1-2-diol and perillic acidinduce T lymphocyte cell death. These data suggest that D-limonene possesses immunomodulatory activ-ity that must be considered when utilizing the compound for therapeutic or commercial purposes.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

D-Limonene is a naturally occurring cyclic terpene that is a ma-jor component of several plant essential oils, including orange,lemon, mandarin, lime and grapefruit. Because of its sweet, citrusfragrance, D-limonene is used widely as an additive in perfumes,soaps, foods and beverages, and is also used as a replacementfor petroleum in cleaning products and paints [1]. In addition topossessing utility as a flavoring and scent agent, D-limonene alsohas been used clinically to dissolve cholesterol-containinggallstones and to relieve gastric acid-induced heartburn [2,3]. Fur-thermore, D-limonene has been shown to possess chemopreventa-tive and chemotherapeutic activity. Treatment with D-limonene,and related monoterpenes, inhibits the formation and develop-ment of mammary carcinomas, lung neoplasms, pancreatictumors, liver cancer, pulmonary adenomas and forestomach tu-mors in rodent models [4–8]. Additionally, phase I and II humanclinical trials of D-limonene and perillyl alcohol, a D-limonenemetabolite, indicate that these agents are well tolerated in cancerpatients and show promise in the treatment of breast and colorec-tal cancers [9–11].

Given the integral role the immune system plays in tumor sur-veillance, it is surprising that only a limited number of studies havebeen conducted investigating the effects of D-limonene on immunesystem (and specifically T lymphocyte) function. Findings from thefew existing studies indicate that D-limonene has immunomodula-

ll rights reserved.

logy, Lebanon Valley College,ates. Fax: +1 (717) 867 6075.

tory properties, although the data is somewhat conflicting.D-Limonene has been observed to increase the total antibody pro-duction, bone marrow cellularity and number of alpha-esterasepositive cells in mice, and both the number and phagocytic activityof alveolar macrophages in rats [12,13]. Additionally, depending onadministration timing and dose administered, D-limonene mayeither suppress or enhance lymphocyte proliferation and specificantibody responses in BALB/c mice [14]. Furthermore, D-limonenetreatment increases nitric oxide production, phagocytic activity,and microbicidal activity of macrophages in lymphoma-bearingmice, while at the same time inhibiting lymphocyte proliferationwhen used at high doses [15]. In vitro studies show that D-limonenesuppresses the lipopolysaccharide-induced production of nitricoxide, prostaglandin E2 and proinflammatory cytokines by RAW264.7 macrophages; whereas perillic acid inhibits the Ras/MAP ki-nase-driven production of IL-2 by purified human T lymphocytes[16,17].

The limited data regarding the effects of D-limonene on immunesystem function suggest that additional research is necessary beforethe therapeutic and/or commercial use of D-limonene progresses.Additionally, because D-limonene is rapidly metabolized to oxygen-ated metabolites, with the predominant circulating metabolites inhumans being perillic acid and limonene-1-,2-diol, it is also impor-tant to investigate the effects of these metabolites on the immunesystem [9,18,19]. In this study we investigated the effects of D-lim-onene on the proinflammatory activities of murine T lymphocytes.We show that D-limonene and its metabolites limonene-1-2-dioland perillic acid inhibit the production by CD3+CD4+ T cells ofIFN-c, IL-2, TNF-a, IL-4 and IL-13, and the production by CD3+CD8+

T cells of IFN-c, IL-2, and TNF-a. Additionally, the upregulation of

Fig. 1. Purification of CD3+CD4+ and CD3+CD8+ T lymphocytes. CD3+CD4+ and CD3+CD8+ T cells were purified via negative selection column. To assess purity, cell surfaceexpression of CD3 and CD4 (A) or CD3 and CD8 (B) were measured by FACS. Dot plots shown are representative of three independent experiments.

C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41 31

CD25, CD69 and CD40L by activated T lymphocytes is modulated byD-limonene, limonene-1-2-diol and perillic acid treatment. Further-more, high concentrations of D-limonene, limonene-1-2-diol andperillic acid induce T lymphocyte cell death. These data suggest thatD-limonene possesses immunomodulatory activity that must beconsidered when utilizing the molecule for therapeutic or commer-cial purposes.

2. Materials and methods

2.1. Mice

8–12 week old female C57BL/6J (wild-type) mice were pur-chased from the Jackson Laboratory (Bar Harbor, ME). All animalstudies were approved by the Lebanon Valley College Animal Careand Use Committee.

2.2. Reagents

D-Limonene (1-methyl-4-(methylethenyl)cyclohexene) waspurchased from Chem Service (West Chester, PA). Limonene-1-2-diol and perillic acid were purchased from Sigma–Aldrich.

2.3. T lymphocyte isolation and activation

Wild-type mice were sacrificed, and the spleens were removed.Splenocytes were passed through a 40-lm nylon cell strainer (BDBiosciences) and collected in PBS. RBC were removed with lysingbuffer (Sigma–Aldrich), and CD3+ T lymphocytes were isolatedwith mouse CD3 column kits, CD3+CD4+ T lymphocytes were iso-lated with mouse CD4 column kits and CD3+CD8+ T lymphocyteswere isolated with mouse CD8 column kits (R&D Systems). Purifiedcells were washed and resuspended in RPMI 1640 medium supple-mented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic (Invitrogen Life Technologies). T cells were activatedby incubation in 96-well plates coated with 2–10 lg/ml immobi-lized anti-CD3 mAb (BD Biosciences) for 24 h (or 6 h for themeasurement of CD69 expression) at 37 �C in 5% CO2. Cells wereco-cultured with 0.5–8 mM D-limonene, limonene-1-2-diol, perillicacid or vehicle control.

2.4. Measurement of cytokine production

IFN-c, IL-2, TNF-a, IL-4 and IL-13 concentrations in superna-tants of T cell cultures were measured by ELISA according to themanufacturer’s protocol (eBioscience).

2.5. Flow cytometry of cell surface markers

Cells were washed and resuspended at 5 � 106 cells/ml in phos-phate buffered saline (PBS). Aliquots (0.1 ml) were placed in iceand labeled for 30 min in the dark with fluorochrome-labelledanti-mouse CD3, anti-mouse CD4, anti-mouse CD8, anti-mouseCD25, anti-mouse CD69, and/or anti-mouse CD40L (eBioscience).Control samples were labeled with isotype-matched control anti-bodies. Stained cells were washed with 1 ml iced PBS and resus-pended in PBS containing 1% paraformaldehyde. The fluorescenceintensity was measured with a dual laser benchtop flow cytometer(FACSCalibur; Becton Dickinson) with a minimum of 20,000 eventsbeing collected. An excitation wavelength of 488 nm and an emissionwavelength of 530 nm were used for FITC-stained cells; an excitationwavelength of 488 nm and an emission wavelength of 585 nm wereused for PE-stained cells; an excitation wavelength of 635 nm and anemission wavelength of 661 nm were used for APC-stained cells.Analysis was performed with Flow Jo software (Tree Star, Inc.).

2.6. Measurement of T cell proliferation

Prior to activation on immobilized anti-CD3 Ab, purified T cellswere stained with 5 lM CFSE. After 24 h, stained cells were washedwith 1 ml iced PBS and resuspended in PBS containing 1% parafor-maldehyde. The fluorescence intensity was measured with a duallaser benchtop flow cytometer (FACSCalibur; Becton Dickinson)with a minimum of 20,000 events being collected. An excitationwavelength of 488 nm and an emission wavelength of 530 nm wereused. Analysis was performed with Flow Jo software (Tree Star, Inc.).

2.7. Measurement of T cell viability

Cells were washed and resuspended at 1 � 106 cells/ml in an-nexin binding buffer. Aliquots (0.1 ml) were labeled for 15 min inthe dark with Alexa Fluor 488 annexin V and propidium iodide.

Fig. 2. D-limonene inhibits TH1 and TH2 cytokine production by purified CD3+ T cells. Purified WT T cells (2,00,000 per well) were incubated on immobilized anti-CD3 mAb inthe presence of varying concentrations of D-limonene or vehicle control. Supernatants were collected after 24 h and IFN-c (A), IL-2 (B), TNF-a (C), IL-4 (D) and IL-13 (E)concentrations were measured by ELISA. Data are shown as the mean ± SEM from three independent experiments performed in quadruplicate. ⁄p < 0.05 vs. vehicle treatedcontrol as assessed by one way ANOVA followed by Dunnett’s multiple comparison test.

Table 1EC50 values of D-limonene to inhibit proinflammatory cytokine production by purifiedCD3+ T cells. Data are the mean ± SEM of three independent experiments performedin quadruplicate.

Mean EC50 (mM ± SEM)

IFN-c IL-2 TNF-a IL-4 IL-32.03 ± 0.35 2.62 ± 0.52 4.22 ± 0.85 2.30 ± 0.29 1.26 ± 0.30

32 C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41

400 lL annexin staining buffer was added to stained cells and thefluorescence intensity was measured with a dual laser benchtopflow cytometer (FACSCalibur; Becton Dickinson) with a minimumof 20,000 events being collected. An excitation wavelength of488 nm and an emission wavelength of 530 nm were used forAlexa Fluor 488-stained cells; an excitation wavelength of488 nm and an emission wavelength of 585 nm were used for pro-

pidium iodide-stained cells. Analysis was performed with Flow Josoftware (Tree Star, Inc.).

2.8. Statistics

Prism software (GraphPad) was used for all statistical analyses.One-way analysis of variance (ANOVA) with post-hoc Dunnett’smultiple comparison was used.

3. Results

3.1. D-Limonene, limonene-1-2-diol and perillic acid inhibitproinflammatory cytokine production by T lymphocytes

T lymphocytes were purified from the spleens of C57BL/6J micevia negative selection column; purification procedures resulted in

Fig. 3. D-limonene, limonene-1-2-diol and perillic acid inhibit proinflammatory cytokine production by CD4+ T cells. Purified CD4+ T cells (2,00,000 per well) were incubatedon immobilized anti-CD3 mAb in the presence of varying concentrations of D-limonene, limonene-1-2-diol, perillic acid or vehicle control. Supernatants were collected after24 h and IFN-c (A), IL-2 (B), TNF-a (C), IL-4 (D) and IL-13 (E) concentrations were measured by ELISA. Data are shown as the mean ± SEM from three independent experimentsperformed in quadruplicate. ⁄p < 0.05 vs. vehicle treated control as assessed by one way ANOVA followed by Dunnett’s multiple comparison test.

C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41 33

>94% pure CD3+ T cell populations, >91% pure CD3+CD4+ T cell pop-ulations and >90% pure CD3+CD8+ T cell populations (Fig. 1). As amodel of TCR-mediated activation, purified murine T lymphocyteswere incubated on immobilized anti-CD3 mAb for 24 h, resultingin the production of the proinflammatory TH1 cytokines IFN-c,IL-2 and TNF-a by pan T cells (CD3+ cells), CD3+CD4+ T cells andCD3+CD8+ T cells and the production of the proinflammatory TH2cytokines IL-4 and IL-13 by CD3+ and CD3+CD4+ T cell populations.The activation of purified pan T cells (CD3+ T cells) in the presenceof D-limonene resulted in a dose-dependent inhibition of TCR-mediated IFN-c (Fig. 2A), IL-2 (Fig. 2B), TNF-a (Fig. 2C), IL-4(Fig. 2D), and IL-13 (Fig. 2E) production. D-Limonene inhibitedIFN-c production by CD3+ T cells with an EC50 value of 2.03 ±0.35 mM; IL-2 production with an EC50 value of 2.62 ± 0.52 mM;

TNF-a production with an EC50 value of 4.22 ± 0.85 mM; IL-4 pro-duction with an EC50 value of 2.30 ± 0.29 mM; and IL-13 produc-tion with an EC50 value of 1.26 ± 0.30 mM (Table 1). Theproduction of TH1 and TH2 cytokines by activated T cells wasinhibited maximally by treatment with 8 mM D-limonene, withIFN-c production inhibited by approximately 97%, IL-2 productioninhibited by 77%, TNF-a production inhibited by 81%, IL-4 produc-tion inhibited by 76% and IL-13 production inhibited by 82%.

To define the effects of D-limonene on specific T cell subsets, aswell as the effects of D-limonene metabolites on T lymphocyte activ-ity, CD4+ and CD8+ T lymphocytes were activated in the presence ofD-limonene, limonene-1-2-diol, perillic acid, or vehicle control. Theactivation of purified CD4+ T cells in the presence of D-limonene,limonene-1-2-diol or perillic acid resulted in dose-dependent

Fig. 4. D-limonene, limonene-1-2-diol and perillic acid inhibit proinflammatory cytokine production by CD8+ T cells. Purified CD8+ T cells (2,00,000 per well) were incubatedon immobilized anti-CD3 mAb in the presence of varying concentrations of D-limonene, limonene-1-2-diol, perillic acid or vehicle control. Supernatants were collected after24 h and IFN-c (A), IL-2 (B) and TNF-a (C) concentrations were measured by ELISA. Data are shown as the mean ± SEM from three independent experiments performed inquadruplicate. ⁄p < 0.05 vs. vehicle treated control as assessed by one way ANOVA followed by Dunnett’s multiple comparison test.

34 C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41

inhibitions of TCR-mediated IFN-c (Fig. 3A), IL-2 (Fig. 3B), TNF-a(Fig. 3C), IL-4 (Fig. 3D), and IL-13 (Fig. 3E) production. Similarly,exposure to D-limonene, limonene-1-2-diol or perillic acid inhibitedthe production of IFN-c (Fig. 4A), IL-2 (Fig. 4B) and TNF-a (Fig. 4C) bypurified CD8+ T lymphocytes. Notably, when compared to theeffects of D-limonene, limonene-1-2-diol and perillic acid more po-tently inhibited proinflammatory cytokine production by both CD4+

and CD8+ T cells.Our control assay conditions did not drive T lymphocyte prolif-

eration (Fig. 5A), and exposure to 0.5–8 mM D-limonene had nosignificant effect on this control, non-proliferative state (Fig. 5B–F) suggesting that the inhibitory effects of D-limonene on TCR-mediated cytokine production are independent from any cell cycleregulatory activity.

3.2. D-Limonene, limonene-1-2-diol and perillic acid modulate theupregulation of activation maker expression by T cells

In addition to stimulating the production of proinflammatorycytokines, the incubation of purified murine T lymphocytes onimmobilized anti-CD3 mAb also triggered the upregulation of cellsurface activation marker expression. Interestingly, treatment withD-limonene differentially inhibited activation marker expressionby pan CD3+ T cells: the expression of CD25 and CD69 by activatedCD3+ T cells was not modulated by exposure to 0.5–8 mM D-limo-nene, however, the expression of CD40L was inhibited significantlyby 2, 4 and 8 mM D-limonene treatment, with CD40L expressionbeing reduced by 29.39 ± 3.18%, 40.18 ± 5.22%, and 44.13 ± 8.42%,respectively (Fig. 6A–C). More significant inhibitory effects by D-

limonene on activation marker expression were observed, how-ever, when murine CD3+ T cells were further purified into CD4+

and CD8+ subsets; additionally, exposure to limonene-1-2-diol orperillic acid was found to reduce the expression of CD25, CD69and CD40L by both CD4+ and CD8+ T cells. The cell surface expres-sion by CD4+ T cells of CD25 was reduced by exposure to D-limo-nene, limonene-1-2-diol and perillic acid by as much as13.81 ± 1.33%, 59.1 ± 1.3% and 65.1 ± 1.21%, respectively (Fig. 7A),the expression of CD69 was reduced by as much as 42.21 ± 3.6%,78.22 ± 3.79% and 80.38 ± 1.67%, respectively (Fig. 7B), and theexpression of CD40L was reduced by as much as 28.45 ± 4.81%,66.5 ± 2.59% and 65.02 ± 4.53%, respectively (Fig. 7C). Furthermore,the cell surface expression by CD8+ T cells of CD25 was reduced byexposure to D-limonene, limonene-1-2-diol and perillic acid by asmuch as 21 ± 3.53%, 71 ± 4.13% and 55.43 ± 4.6%, respectively(Fig. 7D). The expression of CD69 by CD8+ T cells was not signifi-cantly reduced by exposure to 0.5–8 mM D-limonene, but wasreduced by up to 76.71 ± 6.97% and 70.03 ± 2.92% by limonene-1-2-diol and perillic acid treatment respectively (Fig. 7E).

3.3. T lymphocyte viability is affected by D-limonene, limonene-1-2-diol and perillic acid treatment

Not only did treatment with D-limonene, limonene-1-2-diol andperillic acid effect the production of proinflammatory cytokines andthe upregulation of CD25, CD69 and CD40L by activated T cells, buthigh doses of D-limonene and its metabolites were found to mark-edly decrease T cell viability. The co-culture of pan T cells, CD4+ Tcells or CD8+ T lymphocytes with 8 mM D-limonene resulted in an

Fig. 5. D-limonene treatment does not modulate T cell proliferation. Purified CD3+ T cells (2,00,000 per well) were stained with CFSE and incubated on immobilized anti-CD3mAb for 24 h in the presence of vehicle (A) or 0.5 mM (B), 1 mM (C), 2 mM (D), 4 mM (E) or 8 mM (F) D-limonene. Cell proliferation was assessed by FACS. Histograms shownare representative of three independent experiments performed in triplicate.

C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41 35

approximate 14 fold increase (Fig. 8), 8 fold increase (Fig. 9) and 8fold increase (Fig. 10) in cell death respectively as compared to the

4.85 ± 0.38% cell death observed in vehicle treated controls. Treat-ment with 0.5, 1, 2 or 4 mM D-limonene had no significant effect

Fig. 6. D-Limonene inhibits CD40L expression by activated CD3+ T cells. Purified CD3+ T cells (2,00,000 per well) were incubated on control, uncoated, plates (unact.), orimmobilized anti-CD3 mAb for 24 h in the presence of vehicle or varying concentrations of D-limonene. Cell surface expression of CD25 (A), CD69 (B) and CD40L (C) wasassessed by FACS. CD3+ T cells were gated on for analysis. Data are shown as the mean ± SEM from three independent experiments performed in quadruplicate. ⁄p < 0.05 vs.vehicle treated control as assessed by one way ANOVA followed by Dunnett’s multiple comparison test.

36 C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41

on CD3+, CD4+ or CD8+ T cell viability (Figs. 8–10). Similarly, expo-sure to 0.5, 1, or 2 mM limonene-1-2-diol or perillic acid had no sig-nificant effect on CD4+ or CD8+ T cell viability, but treatment withhigher doses markedly increased cell death. Exposure to 4 and8 mM limonene-1-2-diol increased CD4+ T cell death by approxi-mately 10 fold, and 16 fold, respectively (Fig. 9), and 8 mM limo-nene-1-2-diol treatment increased CD8+ T cell death byapproximately 15 fold (lower doses had no significant effect onCD8+ T cell viability) (Fig. 10). Exposure to 4 and 8 mM perillic acidincreased CD4+ T cell death by approximately 11 fold, and 21 fold,respectively (Fig. 9), and treatment with 4 mM or 8 mM perillic in-creased CD8+ T cell death by approximately 6 fold and 20 foldrespectively (Fig. 10). Notably, D-limonene, limonene-1-2-diol andperillic acid inhibited proinflammatory cytokine production andactivation marker expression by CD4+ and CD8+ T cells at concentra-tions lower than those which resulted in increased cell death, sug-gesting that these anti-inflammatory effects are not a result ofcytotoxicity.

4. Discussion

D-Limonene is currently used widely as a flavoring and scentagent, and holds promise as a pharmaceutical tool for the treatmentand/or prevention of cancer. However, the effects of D-limonene onimmune system function are largely unknown, with several reportspresenting conflicting data. Separate studies have suggested that D-limonene enhances or inhibits the proinflammatory activity of mac-

rophages, and enhances or suppresses the proliferation of lympho-cytes [14,15,17]. Such contradictory results may be due tovariations in the in vivo model systems utilized and/or the dosingregimens of D-limonene employed. However, the safe and effica-cious clinical and commercial use of D-limonene is dependent upona better characterization of its immunomodulatory activity. Tomore fully elucidate the effects of D-limonene exposure on T lym-phocyte activity we utilized an isolated in vitro assay system. Weshow that the TCR-mediated production of both TH1 and TH2 cyto-kines by CD4+ and CD8+ cells is markedly inhibited by D-limonenetreatment, with absolute amounts of cytokines produced beinginhibited by 77-97%. Furthermore, two metabolites of D-limonene,limonene-1-2-diol and perillic acid, also potently suppress the pro-duction of proinflammatory cytokines by both CD4+ and CD8+ Tcells. These data are significant because they suggest that thein vivo administration of D-limonene may have immunosuppressiveeffects that directly or indirectly modulate both T cell-mediated andhumoral immune responses. Although a previous study has shownthat D-limonene treatment increases the total antibody productionin rats, this enhanced response is possibly a byproduct of the in-creased total white blood cell count that was also observed [13].Our data suggest that, in fact, by inhibiting the production of TH2cytokines, such as IL-4, D-limonene may impede the proliferationand differentiation of B lymphocytes, as well as inhibit Ab affinitymaturation and class switching, resulting in an impaired humoralresponse; this warrants further investigation in a future study.Additionally, the inhibition of the TH1 cytokines IFN-c and IL-2, aswell as the inhibition of CD25 expression (a cell surface molecule

Fig. 7. D-limonene, limonene-1-2-diol and perillic acid inhibit activation marker expression by activated CD4+ and CD8+ T cells. Purified CD4+ T cells (A-C) or CD8+ T cells (D-E) (2,00,000 per well) were incubated on control, uncoated, plates (unactivated), or immobilized anti-CD3 mAb for 6 (B and E) or 24 hours in the presence of vehicle or varyingconcentrations of D-limonene, limonene-1-2-diol, or perillic acid. Cell surface expression of CD25 (A and D), CD69 (B and E) and CD40L (C) was assessed by FACS. Data areshown as the mean ± SEM from three independent experiments performed in quadruplicate. ⁄p < 0.05 vs. vehicle treated control as assessed by one way ANOVA followed byDunnett’s multiple comparison test.

C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41 37

necessary for the clonal expansion of T cells), by D-limonene and itsmetabolites may potentially impair the activity of cytotoxic T cells,which are involved in immune surveillance. Previous studies havefound that treatment with D-limonene elicits an increased occur-rence of tubular cell hyperplasia, adenomas and adenocarcinomasof the kidney in male rats [20,21]. Although the mechanisms bywhich D-limonene promotes carcinogenicity remain unknown, itis possible that the suppression of T cell activity may be involved.The inhibition by D-limonene, limonene-1-2-diol and perillic acid

of IFN-c production may also result in an altered interaction be-tween cells of the innate and adaptive immune systems – a phe-nomenon that would be exacerbated by the impairedupregulation of CD40L by T cells activated in the presence of D-lim-onene or its metabolites. Taken together, our data show that D-lim-onene markedly inhibits the proinflammatory activities of Tlymphocytes, and has the potential to modulate the interactionsof T cells with other white blood cells. It has recently been reportedthat D-limonene is an agonist for the adenosine A2A receptor – a

Fig. 8. D-Limonene modulates CD3+ T cell viability. Purified CD3+ T cells (2,00,000 per well) were incubated on immobilized anti-CD3 mAb for 24 h in the presence of vehicle(A) or 0.5 mM (B), 1 mM (C), 2 mM (D), 4 mM (E) or 8 mM (F) D-limonene. Cell viability was assessed via staining with Alexa Fluor 488 annexin V and propidium iodide. Dotplots shown are representative of three independent experiments performed in triplicate.

38 C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41

receptor whose activation has widespread anti-inflammatoryeffects, including the inhibition of proinflammatory cytokine pro-duction, activation marker expression and proliferation of T lym-phocytes [22]. Interestingly, we found that the inhibitory effectsof D-limonene on activated T cell activity cannot be blocked by theselective adenosine A2A receptor antagonist, ZM241385 (data not

shown). We therefore conclude that D-limonene possesses anti-inflammatory activity that is independent from adenosine A2A

receptor activation.Listed in the Code of Federal Regulations as ‘‘generally recog-

nized as safe’’ (GRAS) for use as a flavoring agent, D-limonene isthought to possess low toxicity [23], however several studies have

Fig. 9. D-limonene, limonene-1-2-diol and perillic acid modulate CD4+ T cell viability. Purified CD4+ T cells (2,00,000 per well) were incubated on immobilized anti-CD3 mAbfor 24 h in the presence of 0.5, 1, 2, 4 or 8 mM D-limonene, limonene-1-2-diol or perillic acid. Cell viability was assessed via staining with Alexa Fluor 488 annexin V andpropidium iodide. Dot plots shown are representative of three independent experiments performed in triplicate.

C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41 39

Fig. 10. D-limonene, limonene-1-2-diol and perillic acid modulate CD8+ T cell viability. Purified CD8+ T cells (2,00,000 per well) were incubated on immobilized anti-CD3 mAbfor 24 h in the presence of 0.5, 1, 2, 4 or 8 mM D-limonene, limonene-1-2-diol or perillic acid. Cell viability was assessed via staining with Alexa Fluor 488 annexin V andpropidium iodide. Dot plots shown are representative of three independent experiments performed in triplicate.

40 C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41

C.M. Lappas, N.T. Lappas / Cellular Immunology 279 (2012) 30–41 41

shown both acute and chronic toxicities. The oral LD50 for D-limo-nene in rodents has been reported to range from 4.4 to 6.6 g/kg,and although the National Toxicology Program reported no signsof compound-related toxicity in rats and mice administered D-lim-onene at doses below 1650 mg/kg/day for three weeks, conflictingstudies have reported that doses as low as 600 mg/kg/day cancause decreased weight gain and death in male rats, with dosesranging from 1200 to 2400 mg/kg/day resulting in lethargy,excessive lacrimation and nephropathy [20,24,25]. Furthermore,it has been demonstrated that mice treated for 28 weeks with1000 mg/kg/day of D-limonene exhibited lower mean body weightand increased incidence of multinucleated hepatocytes and cyto-megaly as compared to vehicle treated controls [20]. In humansafety studies, it has been observed that 15 g/day of D-limoneneis the maximum tolerated oral dose, with nausea, vomiting, diar-rhea and slight fatigue being the only observed side effects, andno abnormalities observed in liver, kidney or pancreatic functions[9,18,26]. We show that D-limonene, at a concentration of 8 mM,is directly cytotoxic to isolated CD4+ and CD8+ T lymphocytes,while lower concentrations have no significant effect on T cell via-bility. Similarly, whereas lower concentrations of limonene-1-2-diol and perillic acid have no significant effect on CD4+ or CD8+ Tcell viability, 4–8 mM concentrations do markedly increase celldeath. These results are not entirely surprising as it has been re-ported that one mechanism responsible for the antitumor activityof D-limonene is its induction of apoptosis in cancer cells [27]. Theinduction of T cell death by D-limonene and its metabolites sug-gests that the therapeutic window of the molecule must be care-fully monitored in order to prevent untoward cytotoxic sideeffects which could further diminish immune responses. It is ofnote, however, that D-limonene significantly inhibits TH1 andTH2 cytokine production, as well as CD25, CD69 and CD40L upreg-ulation by activated T cells at concentrations that have no effect oncell viability, indicating that these anti-inflammatory effects arenot merely a byproduct of cytotoxicity. Although dietary intakeof D-limonene varies widely by individual, the average daily per ca-pita D-limonene consumption in the U.S. is estimated to be 16.2 mg[28]. Orally administered D-limonene is almost completely ab-sorbed in the gastrointestinal tracts of both humans and animals,and is rapidly distributed to various tissues in the body, includingthe blood serum, liver, lungs and kidneys, with notably higher con-centrations found in adipose tissue and mammary glands [29–31].The prevalence of limonene as a flavoring and scent agent, as wellas the emergence of the compound as a promising therapeuticagent in the treatment and prevention of cancer necessitates thethorough examination of its immunomodulatory properties, andunderscores the significance of our findings, which show that D-limonene modulates T cell activity and viability.

We conclude that D-limonene, limonene-1-2-diol and perillicacid significantly inhibit the proinflammatory activities of bothCD4+ and CD8+ T lymphocytes and possess cytotoxic potential.These immunosuppressive effects must be considered when evalu-ating therapeutic and commercial applications of D-limonene.

References

[1] Limonene monograph, Cri. Rev. Food Sci. Nutr. 39 (1999) 260–265.[2] H. Igimi, T. Hisatsugu, M. Nishimura, The use of D-limonene preparation as a

dissolving agent of gallstones, Am. J. Dig. Dis. 21 (1976) 926–939.[3] J. Wilkins Jr., Method for treating gastrointestinal disorder, 2002, (US Patent

642045).[4] J.A. Elegbede, C.E. Elson, A. Qureshi, M.A. Tanner, M.N. Gould, Inhibition of

DMBA-induced mammary cancer by the monoterpene D-limonene,Carcinogenesis 5 (1984) 661–664.

[5] T.H. Maltzman, L.M. Hurt, C.E. Elson, M.A. Tanner, M.N. Gould, The preventionof nitrosomethylurea-induced mammary tumors by D-limonene and orangeoil, Carcinogenesis 10 (1989) 781–783.

[6] L.W. Wattenberg, V.L. Sparnins, G. Barany, Inhibition of N-nitrosodiethylaminecarcinogenesis in mice by naturally occurring organosulfur compounds andmonoterpenes, Cancer Res. 49 (1989) 2689–2692.

[7] L.W. Wattenberg, J.B. Coccia, Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone carcinogenesis in mice by D-limonene and citrus fruitoils, Carcinogenesis 12 (1991) 115–117.

[8] P.L. Crowell, A.A. Siar, Y.D. Burke, Antitumorigenic effects of limonene andperillyl alcohol against pancreatic and breast cancer, Adv. Exp. Med. Biol. 401(1996) 131–136.

[9] D.M. Vigushin, G.K. Poon, A. Boddy, J. English, G.W. Halbert, C. Pagonis, M.Jarman, R.C. Coombes, Phase I and pharmacokinetic study of D-limonene inpatients with advanced cancer. Cancer research campaign phase I/II clinicaltrials committee, Cancer Chemother. Pharmacol. 42 (1998) 111–117.

[10] H.H. Bailey, D. Levy, L.S. Harris, J.C. Schink, F. Foss, P. Beatty, S. Wadler, A phaseII trial of daily perillyl alcohol in patients with advanced ovarian cancer:Eastern Cooperative Oncology Group Study E2E96, Gynecol. Oncol. 85 (2002)464–468.

[11] S.M. Meadows, D. Mulkerin, J. Berlin, H. Bailey, J. Kolesar, D. Warren, J.P.Thomas, Phase II trial of perillyl alcohol in patients with metastatic colorectalcancer, Int. J. Gastrointest. Cancer 32 (2002) 125–128.

[12] M. Hamada, K. Uezu, J. Matsushita, S. Yamamoto, Y. Kishino, Distribution andimmune responses resulting from oral administration of D-limonene in rats, J.Nutr. Sci. Vitaminol. (Tokyo) 48 (2002) 155–160.

[13] T.J. Raphael, G. Kuttan, Immunomodulatory activity of naturally occurringmonoterpenes carvone, limonene, and perillic acid, Immunopharmacol.Immunotoxicol. 25 (2003) 285–294.

[14] D.L. Evans, D.M. Miller, K.L. Jacobsen, P.B. Bush, Modulation of immuneresponses in mice by D-limonene, J. Toxicol. Environ. Health 20 (1987) 51–66.

[15] S. Del Toro-Arreola, E. Flores-Torales, C. Torres-Lozano, A. Del Toro-Arreola, K.Tostado-Pelayo, M. Guadalupe Ramirez-Duenas, A. Daneri-Navarro, Effect of D-limonene on immune response in BALB/c mice with lymphoma, Int.Immunopharmacol. 5 (2005) 829–838.

[16] S. Schulz, D. Reinhold, H. Schmidt, S. Ansorge, V. Hollt, Perillic acid inhibits Ras/MAP kinase-driven IL-2 production in human T lymphocytes, Biochem.Biophys. Res. Commun. 241 (1997) 720–725.

[17] W.J. Yoon, N.H. Lee, C.G. Hyun, Limonene suppresses lipopolysaccharide-induced production of nitric oxide, prostaglandin E2, and pro-inflammatorycytokines in RAW 264.7 macrophages, J. Oleo. Sci. 59 (2010) 415–421.

[18] P.L. Crowell, C.E. Elson, H.H. Bailey, A. Elegbede, J.D. Haag, M.N. Gould, Humanmetabolism of the experimental cancer therapeutic agent D-limonene, CancerChemother. Pharmacol. 35 (1994) 31–37.

[19] G.K. Poon, D. Vigushin, L.J. Griggs, M.G. Rowlands, R.C. Coombes, M. Jarman,Identification and characterization of limonene metabolites in patients withadvanced cancer by liquid chromatography/mass spectrometry, Drug Metab.Dispos. 24 (1996) 565–571.

[20] National Toxicology Program, Toxicology and Carcinogenesis Studies of D-Limonene in F344/N Rats and B6C3F1 Mice, NIH Publication Number 90-2802,US Government Printing Office, 2007.

[21] S.D. Turner, H. Tinwell, W. Piegorsch, P. Schmezer, J. Ashby, The male ratcarcinogens limonene and sodium saccharin are not mutagenic to male BigBlue rats, Mutagenesis 16 (2001) 329–332.

[22] H.M. Park, J.H. Lee, J. Yaoyao, H.J. Jun, S.J. Lee, Limonene, a natural cyclicterpene, is an agonistic ligand for adenosine A(2A) receptors, Biochem.Biophys. Res. Commun. 404 (2011) 345–348.

[23] The Unites States Code of Federal Regulations, Title 21, Part 182.60, 2011.[24] World Health Organization, International Agency for Research on Cancer, D-

Limonene, IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 73 (1999) 307–327.[25] J. Whysner, G.M. Williams, D-Limonene mechanistic data and risk assessment:

absolute species-specific cytotoxicity, enhanced cell proliferation, and tumorpromotion, Pharmacol. Ther. 71 (1996) 127–136.

[26] H. Igimi, D. Watanabe, F. Yamamoto, S. Asakawa, K. Toraishi, H. Shimura, Auseful cholesterol solvent for medical dissolution of gallstones, Gastroenterol.Jpn. 27 (1992) 536–545.

[27] J.J. Mills, R.S. Chari, I.J. Boyer, M.N. Gould, R.L. Jirtle, Induction of apoptosis inliver tumors by the monoterpene perillyl alcohol, Cancer Res. 55 (1995) 979–983.

[28] Flavor and Extract Manufacturers’ Association, D-Limonene Monograph, Flavorand Extract Manufacturers’ Association, 1994.

[29] P.L. Crowell, S. Lin, E. Vedejs, M.N. Gould, Identification of metabolites of theantitumor agent D-limonene capable of inhibiting protein isoprenylation andcell growth, Cancer Chemother. Pharmacol. 31 (1992) 205–212.

[30] H. Igimi, M. Nishimura, R. Kodama, H. Ide, Studies on the metabolism of D-limonene (p-mentha-1,8-diene). I. The absorption, distribution and excretionof D-limonene in rats, Xenobiotica 4 (1974) 77–84.

[31] R. Kodama, T. Yano, K. Furukawa, K. Noda, H. Ide, Studies on the metabolism ofD-limonene (p-mentha-1,8-diene). IV. Isolation and characterization of newmetabolites and species differences in metabolism, Xenobiotica 6 (1976) 377–389.