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The Journal of Immunology, 2001, 167: 3454-3462.
Copyright © 2001 by The American Association of Immunologists

Curcumin Inhibits Activation of V{gamma}9V{delta}2 T Cells by Phosphoantigens and Induces Apoptosis Involving Apoptosis-Inducing Factor and Large Scale DNA Fragmentation1

Barbara Cipriani2,*,{dagger}, Giovanna Borsellino{dagger}, Heather Knowles*, Daniela Tramonti{dagger}, Fabio Cavaliere{ddagger}, Giorgio Bernardi§, Luca Battistini{dagger} and Celia F. Brosnan*

* Departments of Pathology and Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461; {dagger} Laboratory of Neuroimmunology and {ddagger} Institute of Neurobiology, Instituto di Ricovero e Cura a Carattere Scientifico Santa Lucia, Rome, Italy; and § Department of Neuroscience, Second University of Rome Tor Vergata, Rome, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Curcumin, in addition to its role as a spice, has been used for centuries to treat inflammatory disorders. Although the mechanism of action remains unclear, it has been shown to inhibit the activation of NF-{kappa}B and AP-1, transcription factors required for induction of many proinflammatory mediators. Due to its low toxicity it is currently under consideration as a broad anti-inflammatory, anti-tumor cell agent. In this study we investigated whether curcumin inhibited the response of {gamma}{delta} T cells to protease-resistant phosphorylated derivatives found in the cell wall of many pathogens. The results showed that curcumin levels >=30 µM profoundly inhibited isopentenyl pyrophosphate-induced release of the chemokines macrophage inflammatory protein-1{alpha} and -1{beta} and RANTES. Curcumin also blocked isopentenyl pyrophosphate-induced activation of NF-{kappa}B and AP-1. Commencing around 16 h, treatment with curcumin lead to the induction of cell death that could not be reversed by APC, IL-15, or IL-2. This cytotoxicity was associated with increased annexin V reactivity, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of apoptosis-inducing factor to the nucleus, and morphological evidence of nuclear disintegration. However, curcumin led to only large scale DNA chromatolysis, as determined by a combination of TUNEL staining and pulse-field and agarose gel electrophoresis, suggesting a predominantly apoptosis-inducing factor-mediated cell death process. We conclude that {gamma}{delta} T cells activated by these ubiquitous Ags are highly sensitive to curcumin, and that this effect may contribute to the anti-inflammatory properties of this compound.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cells that express the {gamma}{delta} TCR represent a unique population of lymphocytes that respond to a range of Ags that are remarkably different from those recognized by {alpha}{beta} TCR lymphocytes. These Ags include nonprocessed proteins; small organic phosphate molecules abundant in bacterial cell walls, particularly mycobacteria; and alkylamines, a class of naturally occurring compounds that are secreted in large quantities by a number of different human pathogens as well as present in certain foods (reviewed in Refs. 1 and 2). The unusual response of {gamma}{delta} T cells is thought to reflect a role for these cells in host defense via the rapid recognition of families of unprocessed Ags with conserved molecular patterns. Consistent with this idea is the observation that these cells significantly expand following infection with a wide range of parasitic, bacterial, and viral agents (reviewed in Ref. 3). However, the widespread distribution of these Ags has also implicated them as targets of the immune response in a number of chronic inflammatory and/or autoimmune conditions (4, 5).

The response of a subset of {gamma}{delta} T cells bearing the V{gamma}9V{delta}2 TCR to nonpeptidic Ags has been shown to be polyclonal and not restricted by MHC class I or class II molecules (1). The nature of these compounds, which have mostly been extracted from mycobacterial lysates, is characterized by the presence of phosphate groups. One of these is isopentenyl pyrophosphate (IPP),3 a 246-Da molecule that has a five-carbon isoprenyl chain and a pyrophosphate moiety (6, 7). Following activation by these compounds, V{gamma}9V{delta}2 cells rapidly secrete proinflammatory cytokines such as TNF-{alpha} and IFN-{gamma} and acquire potent cytotoxic activity, implicating these cells as important mediators of inflammation at sites of Ag recognition (8). Recently, we showed that IPP-induced activation of V{gamma}9V{delta}2+ T cells also resulted in the rapid release of chemokines such as macrophage inflammatory protein (MIP)-1{alpha}, MIP-1{beta}, and RANTES and regulated chemokine receptor expression (9). The induction of these genes is known to require the simultaneous binding of multiple transcription factors, and a central role for NF-{kappa}B in this process has been extensively documented (10, 11, 12), making regulation of this transcription factor a prime target for therapeutic intervention in a number of different inflammatory conditions (13). Recent studies have demonstrated that many ancient remedies, such as curcumin and aspirin, inhibit activation of the NF-{kappa}B signaling cascade (14, 15).

Curcumin is a major constituent of turmeric powder and is extracted from the rhizomes of the plant Curcuma longa L found in southern Asia. In addition to its role as a spice, curcumin has also been used for centuries to treat inflammatory disorders (16). Although the exact mechanism of action for curcumin is not well understood, it has been shown to prevent inhibitory factor I{kappa}B degradation, retaining NF-{kappa}B in the cytoplasm in its inactive form and to inhibit activation of c-Jun N-terminal kinase (17, 18, 19). In addition to its anti-inflammatory properties, curcumin induces the growth arrest and apoptosis of a number of different tumor cell lines, also possibly through an interaction with the NF-{kappa}B signaling pathway (18, 19, 20). Because of its relatively nontoxic properties, curcumin is currently under consideration as an anti-inflammatory agent for intestinal inflammation and tumor therapy (21, 22). In this study we have examined whether the responses of {gamma}{delta} T cells to phosphate Ags are also modulated by curcumin. The data show that curcumin potently inhibits IPP-induced NF-{kappa}B activation, proliferation, and chemokine production by V{gamma}9V{delta}2+ T cells and rapidly induces cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparation and stimulation

PBMCs from healthy donors were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia, Uppsala, Sweden) and cultured at 106 cells/ml in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 20 mM HEPES, and 10 U/ml penicillin and streptomycin (Life Technologies, Grand Island, NY). A total of 10 {gamma}{delta} T cell lines established from nine healthy donors were used in the course of these studies. The use of human tissues was approved by the committee on clinical investigation of the Albert Einstein College of Medicine. Long-term cultures of V{delta}2+ cells were established by stimulating with IPP (Sigma, St. Louis, MO) at 30 µM/ml and were maintained with 50 U/ml recombinant human IL-2 (National Cancer Institute, Frederick, MD). Expression of V{delta}2+ cells was determined by FACS using PE-conjugated B6 mAb (BD PharMingen, San Diego, CA), and lines were used for experimentation when V{delta}2+ T cells represented 90% of the total leukocyte population. At the time of testing V{delta}2+ T cells were restimulated with 30 µM/ml IPP (IPP2X) in the presence of 50 U/ml IL-2. In some cultures IL-15 (50 ng/ml; PeproTech, Rocky Hill, NJ) or TNF-{alpha} (5 ng/ml; Genzyme, Cambridge, MA), cycloheximide (CHX; 2 µg/ml; Sigma), NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC; Sigma), the p38 mitogen-activated protein kinase inhibitor SB 203580 (Calbiochem, San Diego, CA), and the mitogen-activated protein kinase kinase 1 inhibitor PD 98059 (New England Biolabs, Beverly, MA) were added as described in the text. All inhibitors were dissolved in DMSO, and cells were pretreated for 1 h. Curcumin (Sigma) was dissolved in ethanol (20 mM), and control cultures were treated with DMSO or ethanol at the same concentrations. The pan-caspase inhibitor Z-VAD.fmk was used at 50 µM (Enzyme Systems Products, Livermore, CA).

Detection of chemokine production by sandwich ELISA

V{delta}2+ T cells were plated in triplicate in 96-well plates at 2 x 105 cells/well, and supernatants were harvested at 24 h poststimulation. Chemokine levels were quantified by sandwich ELISA using matched Ab pairs as described previously (9). TNF-{alpha} levels were determined using a kit (R&D Systems, Minneapolis, MN).

EMSA

V{delta}2+ T cells (5 x 106) were stimulated in 24-well plates for 1 h with medium alone or 50 U/ml IL-2, IPP2X, or IPP2X in the presence of curcumin at 30 µM. Nuclear extracts were prepared by a modified Dignam method on ice, and EMSA was performed as described previously (23). Oligonucleotides containing the NF-{kappa}B consensus binding sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') or the AP-1 consensus binding sequence (5'-CGC TGG ATG AGT CAG CCG GAA-3') were radiolabeled with [{gamma}-32P]ATP using polynucleotide T4 kinase (Gel Shift Assay Core System kit; Promega, Madison, WI).

Proliferation assay

V{delta}2+ T cell lines were plated in duplicate at 2 x 105 cells/well in 96-well plates. After 22 h cells were pulsed with [3H]thymidine at 0.5 µCi/well, harvested at 28 h, and counted using a 1450 beta liquid scintillation counter (Wallac Triluxe, Gaithersburg, MD).

Flow cytometry

For propidium iodide (PI) staining cells were washed twice in cold PBS, then resuspended in 500 µl PBS, one drop of the vital dye PI was added, and cells were analyzed within 1 h (10,000 events, using CellQuest software, BD Biosciences, Franklin Lakes, NJ). Because curcumin altered the fluorescent profiles of the treated population, quadrants for negative staining were established by treating normal cells with curcumin (30 µM), washing the cells immediately in 1x PBS, and acquiring FACS data. To quantitate cells undergoing apoptosis the PI/annexin V staining kit (BD PharMingen) was used. Cells were stimulated for 16 h, harvested in cold PBS, washed, and stained with 5 µl of Annexin VFITC and 10 µl of PI solution in 100 µl of 1x binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, and 25 µM CaCl2) for 15 min at room temperature in the dark, diluted in 400 µl 1x binding buffer, and immediately subjected to FACS analysis (10,000 events/sample).

Conventional DNA electrophoresis

V{delta}2+ T cells (3 x 106) were lysed in 1 ml Tris (pH 8.0; 10 mM/l), NaCl (100 mM/l), EDTA (25 mM/l), 0.5% SDS, and 1 mg/ml proteinase K (Sigma) overnight at 37°C; precipitated in NaCl (1.2 M); centrifuged 10,000 x g for 30 min; and extracted with phenol-chloroform, and genomic DNA was precipitated with isopropanol. Samples were washed in 70% ethanol, resuspended in 10 mM Tris (pH 8.0)/1.0 mM EDTA, and treated with RNase A for 30 min at 37°C. Equal quantities of each sample (20–30 µg) were electrophoresed on 1.4% agarose gels containing 0.5 mg/ml ethidium bromide plus 1 kbp m.w. markers (Life Technologies).

Pulse-field gel electrophoresis

Cell pellets containing 25 x 106 V{delta}2+ cells/sample were resuspended in 315 µl cell suspension buffer (10 mM Tris (pH 7.2), 20 mM NaCl, and 50 mM EDTA) and 185 µl 2% low melt agarose, and solidified at 4°C for 30 min. Plugs were digested overnight at 55°C in 5 ml of digestion buffer (10 mM Tris-HCl (pH 9.5), 0.5 M EDTA, 1% laurylsarcosine, and 1 mg/ml proteinase K), washed four times in 50 ml of washing buffer (20 mM Tris-HCl (pH 8) and 50 mM EDTA), loaded into a 1% agarose gel, and run in 1x Tris acetate EDTA buffer for 22 h using the CHEF-DR III electrophoresis cell (Bio-Rad, Hercules, CA). Running conditions were optimized to separate DNA fragments of 50–1000 kbp as follows: 12°C temperature, 50–90 s switch time, 120o angle, and 6 V/cm voltage gradient. The standard size DNA {lambda} ladder was included. After staining with 0.5 µg/ml ethidium bromide, bands were visualized using a UV transilluminator (254–360 nm).

Immunocytochemistry and digital imaging

V{delta}2+ T cells (5 x 105) were washed and adhered for 30 min to poly-L-lysine-coated slides, fixed with 4% paraformaldehyde for 30 min, washed, and blocked in 10% normal goat serum/0.4% Triton X/PBS for 1 h at room temperature. Cells were stained for 1 h at room temperature with primary Ab in 2% normal goat serum/0.4% Triton X/PBS. After washing twice in PBS/0.2% Tween 20, secondary Abs (Cappel, Durham, NC) were applied for 30 min at room temperature, and cells were counterstained for 15 min at room temperature with 1 µg/ml 4',6'-diamidino-2-phenylindole dihydrochloride hydrate/PBS solution (Sigma) and mounted in aqueous mounting medium (Gel Mount; Biomeda, Foster City, CA). Fluorescent microscopy was performed on an Olympus IX70 (Melville, NY) with x60 N.A. 1.4 infinity corrected optics. Images were collected with a Photometrics (Tucson, AZ) cooled CCD camera with a KAF 1400 chip using I.P. Lab Spectrum (Scanalytics, Fairfax, VA) on a Power Macintosh. Primary Abs used in this study were CM1 (provided by Idun Pharmaceuticals, San Diego, CA), which targets the active form of caspase 3 (24), anti-poly(ADP-ribose)polymerase (anti-PARP) p85 fragment (Promega, Madison WI), and apoptosis-inducing factor (AIF; Santa Cruz Biotechnology, Santa Cruz, CA). TUNEL assay was performed before PARP staining using the In Situ Cell Death Detection kit (fluorescein conjugated; Roche, Indianapolis, IN).

Assay of extracellular lactate dehydrogenase (LDH) activity

Supernatants were collected in duplicate following 18-h treatment from 106 {gamma}{delta} T cells, and LDH release was determined using a kit according to the manufacturer’s instructions (CytoTox 96; Promega).

Western blot analysis of cytochrome c release

Cells (1.4 x 106) were lysed in a sucrose-containing lysis buffer (25 mM sucrose, 1 mM EDTA, 10 mM Tris (pH 7.5), 0.5 M PMSF, and 10 g/ml leupeptin) for 30 min at 0°C. The lysate was centrifuged for 10 min at 4°C (500 x g), and the supernatant was then centrifuged for 10 min at 4°C (10,000 x g). Cytosolic protein (30 µg) from the supernatant of each experimental condition was subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. Blots were probed for 2 h at room temperature with an Ab to cytochrome c (1/200, clone H-104; Santa Cruz Biotechnology), followed by HRP-coupled secondary Ab (1/15,000; Santa Cruz) and analyzed using an ECL system (Santa Cruz Biotechnology). Quantification was performed using a Kodak Image Station (KDS IS440CF 1.1; Eastman Kodak, Rochester, NY).

Data analysis

Results are expressed as the mean ± SEM. Statistical analysis was performed using Prism software (Prism Software, Lake Forest, CA) and was calculated using ANOVA. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine whether curcumin inhibited the expression and release of proinflammatory mediators by {gamma}{delta} T cells activated with phosphate Ags, we first established T cell lines enriched for V{gamma}9V{delta}2 by culturing PBMCs from three healthy donors in the presence of 30 µM IPP and 50 U/ml IL-2 for 3 wk. At the end of this period V{gamma}9V{delta}2+ T cells constituted >=90% of the total lymphocyte population. The cells were then activated again with IPP (IPP2X, 30 µM) in the presence or the absence of curcumin at 100, 75, 30, 10, and 1 µM, and the release of the chemokines MIP-1{beta}, MIP-1{alpha}, and RANTES was determined by ELISA 24 h poststimulation. As shown in Fig. 1Go, stimulation with IPP resulted in the release of high levels of all three chemokines, with the most robust response found for MIP-1{beta}, in agreement with our previous data (9). The presence of curcumin at levels >=30 µM in the medium potently inhibited the release of all three chemokines from these cells (data for 100 µM not shown).



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  		FIGURE 1. Phosphoantigen-induced chemokine production in {gamma}{delta} T cells is inhibited by curcumin in a dose-dependent fashion. V{delta}2+ T cells were cultured with IL-2 alone or with IPP2X in the presence or absence of varying concentrations of curcumin (75 (C75), 30 (C30), 10 (C10), or 1 (C1) µM) for 24 h. The release of the chemokines MIP-1{beta}, MIP-1{alpha}, and RANTES was determined by ELISA. Data shown are the mean ± SEM of values obtained from three different cell lines derived from three different donors. Asterisks denote values significantly different from the IPP2X-treated cultures: ***, p < 0.01.

 
To investigate both the kinetics of this response and to more clearly establish the least effective dose of curcumin that inhibited chemokine release, three additional V{delta}2+ T cell lines were established from three donors, and chemokine release was assessed at 6, 18, and 24 h poststimulation with IPP2X and IL-2 in the presence of 15, 20, or 25 µM curcumin. For MIP-1{beta}, the percent inhibition of chemokine release from the controls was 43, 45, and 55% at 6 h; 48, 47, and 61% at 18 h; and 13, 23, and 54% at 24 h for 15, 20, and 25 µM curcumin, respectively. Similarly, for MIP-1{alpha} the percent inhibition over control values was 38, 39, and 53% at 6 h; 29, 31, and 49% at 18 h; and 18, 31, and 44% at 24 h for 15, 20, and 25 µM curcumin, respectively. These data show that curcumin rapidly inhibits chemokine production induced by IPP in {gamma}{delta} T cells, but that at low doses (<15 µM) some recovery from the inhibitory effects of curcumin occurs over time. Because IPP also potently induces cytokine production in these cells, the same supernatants were analyzed for TNF-{alpha}. The percent inhibition was 76, 80, and 90% at 6 h; 74, 75, and 85% at 18 h; and 42, 59, and 71% at 24 h for 15, 20, and 25 µM curcumin, respectively. These results indicate that curcumin exerted an even more potent inhibitory effect on TNF-{alpha} release than was noted for the chemokines.

In most cell types the transcription factor NF-{kappa}B plays a critical role in the regulation of proinflammatory gene expression, including chemokines (10, 11, 12). In some cell types curcumin has been shown to inhibit NF-{kappa}B activation at a site upstream of the NF-{kappa}B-inducing kinase I{kappa}B kinase complex (17). However, whether IPP activates the NF-{kappa}B signaling cascade in {gamma}{delta} T cells has not been investigated. To test for this we performed an EMSA using a probe containing a NF-{kappa}B binding sequence (Fig. 2GoA, upper panel). In nuclear extracts derived from control untreated V{delta}2 T cell lines, two mobility shift complexes were observed that could be competed out with a 50-fold molar excess of cold competitor oligonucleotide containing the NF-{kappa}B binding sequence but not with a nonspecific cold competitor oligonucleotide matched for size and guanine cytosine content with the specific probe. When the cells were stimulated again for 1 h with IPP (IPP2X) a striking enhancement in the upper shift complex was observed that was down-regulated by pretreatment with curcumin, whereas pretreatment with curcumin had little effect in the control cultures. Similarly, restimulation with IPP led to the enhancement of binding of the AP-1 to the specific DNA consensus sequence, which was down-regulated by pretreatment of the cultures with curcumin (Fig. 2GoA, lower panel). Interestingly, curcumin also down-regulated AP-1 DNA binding in the control cultures.



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  		FIGURE 2. Phosphoantigen restimulation of {gamma}{delta} T cells leads to activation of NF-{kappa}B and AP-1 that is inhibited by curcumin, and NF-{kappa}B inhibition blocks chemokine production. A, Nuclear extracts were prepared from V{delta}2+ T cells that had been cultured for 1 h in IL-2 (lanes 1–3), IL-2 plus curcumin (lane 4), IPP2X (lane 5), or IPP2X plus curcumin (lane 6). EMSA was performed using a radiolabeled oligonucleotide containing the NF-{kappa}B binding site (upper panel) or the AP-1 binding site (lower panel) or in the presence of specific (lane 2, labeled sc) or nonspecific (lane 3, labeled nsc) competitor oligonucleotides as described in Materials and Methods. Data shown are representative of two independent experiments using cell lines isolated from two different donors. B, V{delta}2+ T cells were restimulated with IPP2X for 24 h in the presence of the NF-{kappa}B inhibitor PDTC (30 µM), and levels of MIP-1{beta} and RANTES in the supernatant were determined by ELISA. Data shown are expressed as the mean ± SEM of values obtained from three different cell lines derived from three different donors. Asterisks denote values significantly different from the IPP2X-treated cultures: ***, p < 0.001; *, p < 0.05.

 
To explore further the role of NF-{kappa}B in IPP-induced chemokine production we tested the effect of the inhibitor of the NF-{kappa}B signaling cascade PDTC (30 µM) on IPP-induced MIP-1{beta} and RANTES at 24 h. The results from three separate donors are shown in Fig. 2GoB. Consistent with the data depicted in Fig. 1Go, IPP2X induced both MIP-1{beta} and RANTES, and in cultures in which the NF-{kappa}B inhibitor PDTC was used, the levels of MIP-1{beta} and RANTES were significantly reduced. Thus, the results further support the conclusion that activation of NF-{kappa}B was required for IPP-induced chemokine expression in {gamma}{delta} T cells.

In addition to its anti-inflammatory properties, curcumin has been shown to cause the growth arrest and death of a number of different tumor cell lines (18, 19, 20, 21, 22). Therefore, we tested the effect of curcumin on the proliferation of {gamma}{delta} T cells following restimulation for 22 h with IPP (Fig. 3Go). In V{gamma}9V{delta}2 T cell lines derived from three different donors curcumin at doses >=30 µM completely inhibited the proliferative response to IPP (p < 0.01). A dose of 10 µM also significantly suppressed IPP-induced stimulation (p < 0.05). Examination of the cell cultures indicated that this effect was associated with a dramatic inhibition of IPP-induced cell aggregation when curcumin was administered at 30 µM (data not shown).



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  		FIGURE 3. Phosphoantigen-induced proliferation of V{delta}2+ T cells is inhibited by curcumin. V{delta}2+ T cells were prepared and restimulated with IPP (IPP2X) alone or in the presence of varying concentrations of curcumin as described in Fig. 1Go. Proliferation was determined by incorporation of [3H]thymidine as described in Materials and Methods. Data shown are the mean ± SEM of three different cell lines derived from three different donors. Asterisks denote values significantly different from control cells cultured in medium alone: *, p < 0.05; **, p < 0.005.

 
To determine whether curcumin was toxic for {gamma}{delta} T cells, unfixed cells were stained with PI to assess cell membrane integrity and were analyzed by FACS. In cells incubated in medium alone ~51% were PI+ following exposure to 30 µM curcumin for 24 h, and in cultures stimulated with IPP and IL-2 (50 U/ml) this increased to about 90%. IPP2X lead to significant toxicity (~50%), which we attribute to Ag-induced cell death, and in these cultures cotreatment with curcumin resulted in 77% toxicity. We then repeated these experiments to determine whether the addition of the T cell growth factor IL-15 (50 ng/ml), which has been shown to enhance {gamma}{delta} T cell responses to Ag (25), or the presence of APC could protect against curcumin-induced cell death. However, no differences in toxicity were detected (data not shown). These results indicate that curcumin is highly toxic for {gamma}{delta} T cells and that this effect is accentuated in {gamma}{delta} T cells that have been activated with high dose IL-2.

To assess whether curcumin-treated {gamma}{delta} T cells were dying via the induction of apoptosis we used a combination of PI and annexin V staining and FACS analysis (26). This procedure distinguishes among cells that are dying from apoptosis, which stain positively for annexin V (used to detect the early apoptotic-associated translocation of phosphatidyl serine from the inner to the outer leaflet of the cell membrane) and negatively for PI (used to assess plasma membrane integrity); cells already dead from apoptosis, which stain positively for both markers; and cells that are dying by necrosis, which stain positively only for PI. Cells treated with TNF-{alpha} (5 ng/ml) in the presence of CHX (2 µg/ml) were used as a positive control for apoptosis. Because restimulation with IPP induced cell death in about 50% of the cells, we studied the mechanism of curcumin-induced toxicity in cells treated with medium alone or with IL-2 alone. At 45 min and 4 h no evidence of either apoptosis or necrosis was observed in curcumin-treated cells incubated in medium alone or in medium containing IL-2. Similarly, in cultures treated with TNF-{alpha} and CHX only ~7% were annexin V+ at these same time points. However, by 16 h significant increases in both PI and annexin V reactivity were observed in cells treated with 30 µM curcumin or with TNF-{alpha} and CHX (data for cells treated with the various combinations in the presence of IL-2 for 16 h are shown in Fig. 4Go). In contrast, no differences from the untreated control cultures were detected in either annexin V reactivity or PI staining in cells treated with 10 µM curcumin (Fig. 4Go). These results suggest that both 30 µM curcumin and TNF-{alpha} plus CHX induce apoptosis in {gamma}{delta} T cells that rapidly progressed to loss of membrane integrity and cell death, as evidenced by PI staining. This conclusion was further supported by analysis of LDH release, which showed that compared with cells treated with IL-2 alone, cells treated with 30 µM curcumin for 18 h demonstrated a 14% increase in LDH release, and an 11% increase was observed in cells treated with TNF-{alpha} and CHX.



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  		FIGURE 4. Treatment of V{delta}2+ T cells with curcumin leads to increased annexin V and PI staining in a dose-dependent fashion. V{delta}2+ T cell lines were cultured for 16 h in the presence of IL-2 alone, IL-2 plus curcumin at 10 or 30 µM, or TNF-{alpha} plus CHX (5 ng/ml and 2 µg/ml, respectively) and subjected to FACS analysis. Gates were determined as described in Materials and Methods. Data shown are representative of two independent experiments with two different cell lines derived from two different donors.

 
However, in some cells annexin V reactivity has been found to be reversible and not necessarily associated with the onset of apoptosis (27). Thus, we explored further the mechanism of curcumin-induced cell death by examining the expression of other apoptotic indicators. Caspase-3/CPP32, a member of the IL-1-onverting enzyme family, is considered an executioner protease in mammalian cells during apoptosis (28). It is present in the cytoplasm as a proenzyme that is activated by proteolytic processing to form the active large (p20) and small (p10) subunits. Using the Ab CM1 that recognizes the p20 subunit of cleaved caspase-3, but not the zymogen (24), increased intranuclear staining was detected in only a few cells that had been treated with 30 µg/ml IPP2X (5%), 30 µM curcumin (8%), or IPP2X plus curcumin (5%) for 4 h (data not shown). However, by 8 h increased immunoreactivity for activated caspase-3 was detected in the nuclear compartment in both IPP2X-treated and curcumin-treated cells (Fig. 5GoA), and quantification of these data indicated significantly increased expression of activated caspase-3 over that in untreated cells for all three groups (Fig. 5GoB). These data suggest that curcumin activates an apoptotic mechanism of cell death, and this conclusion was further supported by evidence that incubation with the pan-caspase inhibitor Z-VAD.fmk significantly inhibited the appearance of cells expressing both annexin V and PI reactivity in cells treated with curcumin (Fig. 5GoC). However, treatment with Z-VAD.fmk did not prevent cell death, because all of the curcumin-treated cells eventually became PI positive.



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  		FIGURE 5. Analysis of activated caspase-3 and effects of the caspase inhibitor Z-VAD.fmk. A, V{delta}2+ T cell lines were treated for 8 h as shown and immunoreacted for activated caspase-3 (green), stained with DAPI (blue), and examined by digital imaging. The size marker is shown on the bottom right panel. B, The percentage of cells expressing intranuclear activated caspase-3 was determined by counting 100 cells/treatment (*, p < 0.01; ***, p < 0.001). C, V{delta}2+ T cell lines were preincubated for 1 h with the pan-caspase inhibitor Z-VAD.fmk (50 µM), followed by incubation with IPP with and without curcumin for the times shown, and the percentage of cells expressing annexin V and PI was determined by FACS (10,000 events analyzed). Data shown are representative of two independent experiments using two different cell lines derived from two different donors.

 
Once activated, caspases initiate a cascade of proteolysis that involves further processing/activation of additional caspases, and ultimately cleavage of specific cellular proteins, such as PARP, an enzyme involved in DNA repair, leading to its inactivation (29). Assessment of immunoreactivity for the cleaved form of PARP (p85 fragment) indicated that this was also increased in cells treated with 30 µg/ml IPP2X, 30 µM curcumin, and IPP2X plus curcumin for 8 h (Fig. 6GoA). Interestingly, however, whereas in cells treated with either IPP2X or TNF-{alpha} plus CHX increased immunoreactivity for the cleaved form of PARP was associated with evidence of DNA strand breaks, as determined by TUNEL staining, this was not detected in the cells treated with curcumin (Fig. 6Go). To pursue this observation further, we assessed DNA ladder formation using conventional 1% agarose DNA gel electrophoresis (Fig. 7GoA). No evidence of DNA fragmentation was obtained following incubation of {gamma}{delta} T cells with 30 µM curcumin for 14, 16, or 24 h (24 h data shown in lane 2), whereas clear DNA ladder formation was observed in {gamma}{delta} T cells cultured for 24 h with IPP2X (lane 1) or for 24 h with TNF-{alpha} plus CHX (lane 3). However, changes in the integrity of DNA are optimally evaluated using pulse-field gel electrophoresis, permitting the detection of high m.w. fragments (30). As shown in Fig. 7GoB, the addition of curcumin to cultures incubated with either IL-2 or IPP2X led to the generation of high m.w. fragments (~50 kb) that did not progress to the formation of internucleosomal DNA laddering (lanes 2 and 4). As noted previously, cells activated either with IL-2 alone or IL-2 plus Ag were more sensitive to the toxic effect of curcumin (compare lanes 2 and 4 with lane 5) than control cultures incubated without IL-2.



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  		FIGURE 6. Analysis of PARP p85 fragment and assessment of DNA strand breaks using the TUNEL assay demonstrate differences in the apoptotic process following treatment with curcumin (C). A, V{delta}2+ T cell lines were incubated for 8 h as shown and then triple stained using an Ab to the p85 PARP cleavage product (red), TdT-mediated dUTP nick end-labeling using the TUNEL assay (green), and the DNA binding dye DAPI (blue). The cells were examined by digital imaging. B, The percentage of cells expressing intranuclear PARP or TUNEL was determined by counting 100 cells/treatment (**, p < 0.005 for IL-2 and curcumin; *, p < 0.001 for IPP and curcumin; PARP vs TUNEL). Data shown are representative of two independent experiments using two different cell lines derived from two different donors.

 


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  		FIGURE 7. Incubation of V{delta}2+ T cell lines with 30 µM curcumin for 24 h results in large scale DNA fragmentation, but not oligonucleosomal DNA fragmentation. A, V{delta}2+ T cell lines were cultured with IL-2 and IPP2X (lane 1), IL-2 and 30 µM curcumin (lane 2), or TNF-{alpha} and CHX (lane 3) for 24 h. DNA was extracted and subjected to horizontal 1.4% agarose gel electrophoresis as described in Materials and Methods. The size of the DNA fragments is indicated on the left. B, V{delta}2+ cells were treated with IL-2 (lane 1), IL-2 plus curcumin (lane 2), IPP2X (lane 3), IPP2X plus curcumin (lane 4), and curcumin alone (lane 5) for 24 h. Cells were embedded in agarose gel and subjected to pulse-field gel electrophoresis. The sizes of the DNA fragments are indicated on the right. Data shown are representative of two independent experiments using two different cell lines isolated from two different donors.

 
The induction of high m.w. DNA fragmentation has been proposed to follow the translocation of AIF from the outer mitochondrial membrane to the nucleus and to correlate with morphological evidences of chromatin condensation (31, 32). Immunofluorescent staining of AIF in control cells incubated with IL-2 or with IPP plus IL-2 demonstrated a punctate staining pattern restricted to the cytoplasm and predominantly localized to the perinuclear region (green, Fig. 8GoA, upper panels). This staining pattern is characteristic for mitochondria (33). However, in cells treated with curcumin for 4 h a shift in the staining pattern was observed, such that diffuse reactivity within the cytoplasm as well as within the nuclear compartment was clearly visible (lower panel). Thus, treatment with curcumin led to the rapid release of AIF from the outer mitochondrial membrane that was not detected in cells treated with IPP2X alone (Fig. 8GoC). To assess whether AIF release was associated with loss of cytochrome c from the mitochondria, Western blot analysis was performed on cytosolic fractions harvested from cells treated for 5 h with IL-2 and 30 µM curcumin, IL-2 and IPP2X, or IL-2, IPP2X, and 30 µM curcumin. No difference in cytochrome c release was detected between cells cultured with IL-2 and those treated with IL-2 and curcumin. However, a clear increase in cytochrome c release was noted in cells treated for the same time period with either IL-2 and IPP2X or IL-2, IPP2X, and curcumin. These data show that cells treated with Ag and IL-2 were more sensitive to the toxic effects of curcumin and support the conclusion that AIF release precedes cytochrome c release in curcumin-treated cells. Ultrastructural studies of cells exposed to curcumin for 12 h demonstrated blistering of the nuclear envelope and partial chromatin condensation (Fig. 8GoB) compared with control cells. This is consistent with the pattern of large scale DNA fragmentation initiated by AIF in vivo and as detected by the pulse-field gel electrophoresis assays described above.



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  		FIGURE 8. Curcumin treatment leads to the nuclear translocation of AIF and nuclear chromatin condensation. A, V{delta}2+ T cell lines were treated with IL-2 (upper left panel), IL-2 and curcumin (lower left panel), IPP2X and IL-2 (IPP; upper right panel), or IPP2X, IL-2, and curcumin (lower right panel) for 4 h. Cells were double stained using an Ab directed to AIF (green) and the DNA binding dye DAPI (blue). B, V{delta}2+ T cells were cultured in IL-2 for 12 h in the presence (C, lower panel) or absence (CTR, upper panel) of curcumin and analyzed ultrastructurally by electron microscopy (magnification, x1500). C, The percentage of cells expressing intranuclear AIF was determined by counting 100 cells/treatment as described in A (**, p < 0.002; *, p < 0.005; treated vs control). D, The presence of cytochrome c in the cytoplasm for the conditions shown in C was determined by Western blotting. The values under each band were determined by densitometry and are expressed in arbitrary units. Data shown are representative of two independent experiments using two different cell lines derived from two different donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we examined the interaction between curcumin and {gamma}{delta} T cells responding to the phosphate Ag IPP. From the data we can make the following conclusions: 1) IPP activated both the NF-{kappa}B and AP-1 signaling cascades in these cells; 2) treatment with curcumin inhibited IPP-induced activation of NF-{kappa}B and AP-1; 3) curcumin also inhibited IPP-induced chemokine release, proliferation, and activation-dependent aggregation; and 4) curcumin demonstrated significant dose- and time-dependent cytotoxic activity toward {gamma}{delta} T cells through activation of a cell death pathway that resulted in nuclear apoptosis and large scale DNA fragmentation. Because the response to this synthetic phosphate Ag is thought to reflect the activation of this subset of T cells to families of unprocessed Ags with conserved molecular patterns (1, 2, 3), the results suggest that these effects of curcumin contribute to the anti-inflammatory properties of this spice.

These studies were initially undertaken as part of an effort to understand the signaling pathways activated by phosphate Ags in {gamma}{delta} T cells. Recognition of these Ags involves the coexpression of both V{gamma}9 and V{delta}2 chains, with the CDR3 loop of the TCR{gamma} chain and adjacent residues playing a crucial role in the recognition process (2, 7). However, the signaling pathways activated by phosphoantigens following interaction with the {gamma}{delta} TCR remain to be elucidated. A role for NF-{kappa}B can be surmised by the demonstration that activation with IPP leads to the rapid induction of large amounts of proinflammatory cytokines and chemokines, all of which contain NF-{kappa}B binding elements in their promoters (8, 9, 10, 11, 12). This conclusion is further supported by the observation that the NF-{kappa}B inhibitor PDTC completely inhibited IPP-induced MIP-1{beta} production and partially inhibited IPP-induced RANTES production.

Using EMSA with oligonucleotides specific for the NF-{kappa}B and AP-1 binding sequences we now show that re-exposure to IPP induces a shift complex within the nuclear extracts within 1 h of stimulation that could be competed out with specific probe, and supershift analysis demonstrated that only Abs to p65/RelA and p50 were active in this assay (B. Cipriani, unpublished observation), indicating that NF-{kappa}B is the conventional p65/p50 heterodimer most commonly induced by inflammatory stimuli (13). Cotreatment with curcumin completely blocked the appearance of these shift complexes in nuclear extracts in IPP-treated cells, in agreement with data from other cell types following activation with cytokines, phorbol esters, and hydrogen peroxide (14, 17). In IL-1{beta}- or TNF-{alpha}-stimulated intestinal epithelial cells, Jobin et al. (14) showed that the inhibitory effect of curcumin occurred upstream of the I{kappa}B kinase complex, resulting in the inhibition of cytokine-induced ICAM-1 and IL-8 expression. Curcumin has also been shown to inhibit c-Jun N-terminal kinase activity (34), suggesting that curcumin targets a common upstream kinase or multiple kinases induced by inflammatory signals. Although we have not addressed sites of interaction of curcumin with the NF-{kappa}B signaling cascade in this study, it is of interest to note that FACS analysis demonstrated that curcumin rapidly associated with components of the cell membrane, suggesting that curcumin could function by interfering with membrane signaling events. A similar conclusion has been reached following studies of the effect of curcumin on erythrocytes, where the authors concluded that curcumin induced apoptosis-independent alterations in membrane dynamics associated with phospholipid scrambling at the plasma and possibly also the mitochondrial cellular membranes (35).

Studies both in vivo and in vitro have shown that curcumin is also inhibitory for a broad range of tumors, including mammary adenocarcinomas, colon carcinomas, 12-O-tetradecanoylphorbol-13-acetate-induced skin tumors in mice, and phorbol-ester induced transformation of murine fibroblasts (18, 19, 36, 37, 38). However, the actual mechanisms involved in this process remain poorly defined. It has been suggested by Pickowa et al. (20) that in Jurkat cells curcumin activates a novel apoptotic pathway that is independent of changes in the mitochondrial transmembrane potential or activation of caspase 3. In V{delta}2+ T cells, curcumin-induced cell death was dose and time dependent, could not be reversed by addition of the T cell growth factors IL-2 and IL-15, and was associated with increased Annexin V reactivity, caspase-3 activation, cleavage of PARP, and translocation of AIF to the nucleus.

Studies both in vivo and in vitro have suggested that there are two redundant parallel pathways that lead to chromatin processing during apoptosis (31). One of these involves translocation of the mitochondrial outer membrane protein AIF, a flavoprotein oxidoreductase, to the nucleus, resulting in peripheral chromatin condensation and large scale DNA fragmentation (31, 32, 33). The other involves the activation of caspases, leading to the generation of caspase-activated DNase, the enzyme responsible for oligonucleosomal DNA fragmentation (28, 39). In V{delta}2+ T cells, treatment with curcumin led to the early release of AIF, partial chromatin condensation, and large scale DNA fragmentation to ~50 kb, yet failed to show oligonucleosomal DNA fragmentation. In contrast, treatment with either IPP2X or TNF and CHX resulted in the formation of a classical DNA ladder, demonstrating that the failure to detect oligonucleosomal DNA fragmentation in response to curcumin was not due to an inherent defect in the apoptotic cascade in these cells. Although it has been proposed that AIF translocation occurs in cells undergoing either apoptosis or necrosis (33), more recent studies using mice in which the gene for AIF has been inactivated strongly support the conclusion that AIF induces an apoptotic pathway that exhibits the classical ultrastructural features of apoptosis in which a peripheral type of chromatin compaction predominated, but without the advanced chromatin condensation typical of caspase-dependent apoptosis (40). From these results we conclude that treatment with curcumin led to an apoptotic death process dominated by the AIF-dependent pathway, whereas in cells treated with either IPP2X or TNF-{alpha} plus CHX the caspase-dependent pathway predominated. Because AIF stimulates apoptosis via an as yet unknown caspase-independent mechanism (31, 41), these data further suggest that the cytotoxic events mediated by curcumin may provide a useful tool in which the processes that contribute to the selective activation of specific apoptotic pathways can be explored.

Given the wide distribution of the Ags recognized by these V{gamma}9V{delta}2+ T cells and the rapidity with which proinflammatory cytokines such as IFN-{gamma} and TNF-{alpha} and chemokines such as MIP-1{alpha} and MIP-1{beta} are produced through pathways that appear to differ from {alpha}{beta} T cells (8, 9, 42), these cells could play an important role in the transition from the innate to the acquired immune response by biasing reactions toward a Th1-type response. This would suggest that at sites of Ag recognition curcumin could effectively inhibit {gamma}{delta} T cell activation, which could have broad implications for the activation of both innate and acquired immune responses. However, it is of interest to note that {gamma}{delta} T cells in the gut, where curcumin levels would be expected to be the highest, preferentially use variable regions V{delta}1 and V{delta}3 rather than V{delta}2. This subset of {gamma}{delta} T cells has also been implicated in a broad response to stress-related proteins (43), and thus it will be important to determine in future studies whether this subset of T cells is also highly sensitive to the anti-inflammatory properties of curcumin.


   Acknowledgments
 
We thank David Gebhard for assistance with the FACS analysis, Drs. Rick Kitsis and Luciano D’Adamio for helpful discussions of apoptosis, and Dr. Bettina Fries for assistance with the CHEF technique and AIF for imaging. We also thank Idun Pharmaceuticals (San Diego, CA) for the CM-1 Ab.


   Footnotes
 
1 This work was supported by Ministero della Sanita’ (Progetto Finalizzato), Ministero dell’Universita’ e della Ricerca Scientifica e Tecnologica, National Multiple Sclerosis Society Grant RG3037A3, and U.S. Public Health Service Grants NS31919 and 5P30CA13330. Back

2 Address correspondence and reprint requests to Dr. Barbara Cipriani, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: cipriani{at}aecom.yu.edu Back

3 Abbreviations used in this paper: IPP, isopentenyl pyrophosphate; IPP2X, restimulated with 30 µg/ml IPP; AIF, apoptosis-inducing factor; CHX, cycloheximide; LDH, lactate dehydrogenase; MIP, macrophage inflammatory protein; PARP, poly(ADP-ribose) polymerase; PDTC, pyrrolidine dithiocarbamate; PI, propidium iodide; DAPI, 4',6'-diamidino-2-phenylindole dihydrochloride hydrate. Back

Received for publication November 27, 2000. Accepted for publication July 9, 2001.


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