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Chalcones from Chinese liquorice inhibit proliferation of T cells and production of cytokines
International Immunopharmacology 2 (2002) 545 – 555 www.elsevier.com/locate/intimp
Chalcones from Chinese liquorice inhibit proliferation of T cells and production of cytokines Lea Barfod a, Ka˚re Kemp b,*, Majbritt Hansen a, Arsalan Kharazmi a a Department of Clinical Microbiology, Centre for Medical Parasitology, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark b Department of Infectious Diseases, Centre for Medical Parasitology, M7641, Copenhagen University Hospital (Rigshospitalet), Tagensvej 20, 2200 Copenhagen N, Denmark
Received 20 August 2001; accepted 2 December 2001
Abstract Licochalcone A (LicA), an oxygenated chalcone, has been shown to inhibit the growth of both parasites and bacteria. In this study, we investigated the effect of LicA and four synthetic analogues on the activity of human peripheral blood mononuclear cell proliferation and cytokine production. Four out of five chalcones tested inhibited the proliferation of lymphocytes measured by thymidine incorporation and by flow cytometry. The production of pro- and anti-inflammatory cytokines from monocytes and T cells was also inhibited by four of five chalcones. Furthermore, intracellular detection of cytokines revealed that the chalcones inhibited the production rather than the release of the cytokines. Taken together, these results indicate that LicA and some analogues may have immunomodulatory effects, and may thus be candidates not only as anti-microbial agents, but also for the treatment of other types of diseases. D 2002 Published by Elsevier Science B.V. Keywords: Chalcones; T cells; Cytokines
1. Introduction The discovery of many new drugs against various diseases has been inspired by the use of plants in traditional medicine [1]. Liquorice has been used in China for the treatment of gastric and duodenal ulcers, bronchial asthma, Addison’s disease, food and drug poisoning and skin diseases such as eczema and urticaria [2]. We have reported that Licochalcone A, an oxygenated chalcone originally isolated from Chinese
*
Corresponding author. Tel.: +45-35-45-73-75; fax: +45-3545-76-44. E-mail address: [email protected] (K. Kemp).
liquorice roots, inhibits the in vitro growth of both chloroquine-susceptible and chloroquine-resistant Plasmodium falciparum and protects mice from lethal P. yoelii infections [3]. Others have supported these findings showing inhibitory effects of chalcone analogues on in vitro growth of P. falciparum [4]. Licochalcone A also inhibits the in vitro growth of Leishmania major and L. donovani promastigotes and amastigotes [5], prevents lesion development in mice infected with L. major and reduces the parasite load in the spleen and liver of hamsters infected with L. donovani [6]. Finally, some synthetic chalcone analogues of LicA have also shown comparable activities to LicA [7,8]. Some anti-protozoan drugs interfere with the immune response. Among others, the most prominent
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anti-malaria drug, chloroquine [9 – 11], inhibits the release of pro-inflammatory cytokines [9]. Since LicA and its analogues are potential antiprotozoan drugs, the effect of these on the human immune system is important. To this end, we have investigated the influence of LicA and synthetic analogues on cytokine production and lymphocyte proliferation.
2. Materials and methods 2.1. Chalcones Licochalcone A (LicA) and 21 synthetic analogues were synthesized at the Department of Medicinal Chemistry, Royal Danish School of Pharmacy (Copenhagen, Denmark). LicA and four of the analogues were selected and included in this study based on the effect on LPS-induced TNF-a secretion. Chalcones were diluted in DMSO and RPMI 1640 to a maximum final concentration of 0.5%. 2.2. Isolation of mononuclear cells and viability Heparinized peripheral blood (20 ml) was collected from healthy Danish donors. Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep (Nyegaard, Oslo, Norway) density centrifugation and washed twice in RPMI 1640. Finally, the cells were dissolved in growth media (RPMI 1640 containing 10% human serum, 40 IU/ml penicillin/streptomycin (Gibco, Paisley, UK) and 1.6 mM L -glutamine (Sigma-Aldrich, Germany) or cryopreserved in liquid nitrogen as described previously [12]. Prior to experiments, frozen PBMCs were rapidly thawed, washed and the viability was determined by trypan blue staining. Incubation with 25 mg/ml of LicA or the analogues PH141, PH138, or PH105 resulted in no less than 93% viability. The cells incubated with 25 mg/ml of PH100 had less than 80% viability. At 12.5 mg/ml, this viability was increased to more than 97%. 2.3. Measurement of lymphocyte proliferation by [3H]-thymidine incorporation Frozen PBMCs were rapidly thawed, washed and the viability was determined by trypan blue staining.
Wells of round-bottomed plates received 100 ml of PBMC (1 106 cells/ml) in growth media. PHA (20 ml) at a final concentration of 40 mg/ml and 20 ml of medium were added to test cultures and control cultures, respectively. Chalcone dilutions were added after incubation for 2 days at 37 C in a humidified atmosphere containing 5% CO 2. The cells were pulsed with 20 ml/well (1.85 Mbq/well) of [3H]thymidine (New England Nuclear, Boston, MA, USA) after incubation for another 2 h at 37 C. Cells were harvested onto glass fibre filters after 20 h and the incorporation of [3H]-thymidine into DNA was determined by a Matrix b-counter (Packard, Greve, Denmark). 2.3.1. Cytokine measurements in culture supernatants Frozen PBMCs were rapidly thawed and washed. The cells were stimulated for 22 h at 37 C with either 10 ng/ml E. coli LPS (Difco, Detroit, MI) or PMA (Sigma-Aldrich) and ionomycin (Calbiochem, USA) (final concentrations of 0.1 and 3.5 mg/ml) and with or without dilutions of the chalcones at a final concentration of 0– 100 mg/ml. The concentrations of IFN-g, TNF-a, IL-1b, IL-6 and IL-10 in the supernatants were measured by ELISA in triplicates (R&D Systems, London, England). TNF-a, IL-1b, IL-6 and IL-10 concentrations were measured following LPS stimulation and IFN-g was measured after PMA and ionomycin stimulation. 2.3.2. Flow cytometry 2.3.2.1. Intracellular cytokine detection. For the measurements of intracellular cytokines in T cells, monensin (1.5 mM; Sigma, St. Louis, MO, USA), ionomycin (1 mM), PMA (50 ng/ml) and chalcone dilutions (25 mg/ml) were added to PBMC cultures in polystyrene tubes for 4 h. Measurement of intracellular cytokines in monocytes was done in the following manner: PBMCs were stimulated 6 h in polystyrene tubes at 37 C with LPS (final concentration of 10 ng/ml), monensin (final concentration of 0.001 mg/ml) (Sigma-Aldrich) and 0– 25 mg/ml of the chalcones. The intracellular staining procedure was based on other studies [13]. In brief, following stimulation, cells were harvested and washed in PBS, resuspended in staining buffer (PBS, 0.5% BSA, 0.01% NaN3) and
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labelled with antibodies directed against the T-cell surface marker CD3 (Dako, Glostrup, Denmark) for 20 min at room temperature. The cells were washed twice in staining buffer, fixed in staining buffer containing 2% formaldehyde (Sigma) and then washed twice in staining buffer and twice in freshly made saponin buffer (staining buffer containing 0.1% (w/v) saponin (Sigma)). Finally, cells were incubated with anti-cytokine (IFN-g or TNF-a, Pharmingen, CA, USA) antibodies for 30 min. Following cytokine labelling, the cells were washed twice in saponin buffer, twice in staining buffer, resuspended and analysed on an EpicsXL-MCL flow-cytometer (Coulter, Miami, FL, USA). Flow cytometric data were analysed by Winlist 3D software (Verity Software House, ME, USA). Samples were live-gated on lymphocytes or monocytes by forward and side scatter gates and limits for fluorescence positive cells were set using nonspecific IgG antibodies.
colour reaction was terminated by the addition of heat-inactivated fetal calf serum (HFCS). The cell suspension was diluted in RPMI 1640 supplemented with 58.4 mg/ml L-glutamine, 20 IU/ml penicillin/ streptomycin (Gibco) and 10% HFCS and washed twice. Finally, the cells were resuspended in growth media (seen previously) at a cell density of 2 106/ ml. The cells were incubated with or without (DMSO alone) 125-ml chalcone dilutions and with or without 50-ml PHA for 3 days. Following incubation, cells were incubated 20 min in formalin buffer and washed twice in PBS supplemented with 10% HFCS. Flow cytometric analyses were performed using Modfit LT 3.0 software (Verity Software House). The unstimulated control samples were used to determine the parent peak and, on this basis, the next generations were defined as the cells having proliferated one to three times. The proliferation index was calculated according to the Modfit LT 3.0 software.
2.3.3. Lymphocyte proliferation Freshly isolated PBMCs were washed twice in PBS and after the last wash, pellet was dissolved in Diluent C (Sigma-Aldrich) and rapidly added the fluorescent membrane due PKH26 solution (1:250 in Diluent C) (Sigma-Aldrich). After 3 min at RT, the
2.4. Statistical analysis All statistical calculations were performed using Sigma Stat software 2.0 (SPSS Science, USA). Initial analyses were one-way ANOVA or Kruskal – Wallis one-way ANOVA on ranks. If the P-values for the
Fig. 1. Plot showing the inhibition of PHA-stimulated lymphocytes by five chalcones. The concentration of DMSO in the control is similar to the concentrations in the chalcone dilutions. The values are mean of three independent triple determinations and error bars indicate the standard deviation from the mean.
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Fig. 2. (a) Representative flow cytometry data showing the PKH26 fluorescence of lymphocytes and the separation of the different populations. Left: the unstimulated control defining the parent peak; middle: PHA-stimulated control; right: LicA and PHA-stimulated lymphocytes. (b) Histogram showing the percentage of lymphocytes in different generations determined by PKH26 staining. The numbers in top of the columns are the proliferation indexes. The values are mean values from four independent determinations with different donors. Standard deviations from the mean are shown by error bars. Asterisks indicate proliferation indexes statistically and significantly lower compared to the stimulated control. The concentration of DMSO in the DMSO control is 0.25%, which is the same as in the highest LicA dilution (25 mg/ml).
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Fig. 3. Graphs showing the inhibition of cytokine secretion from LPS-stimulated (A – D) or PMA- and ionomycin-stimulated (E) PBMC by five chalcones. Values are mean of three to six independent triple determinations with different donors. Error bars define the standard deviation.
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Fig. 4. (a) Plots showing the frequency of monocytes expressing TNF-a after 6 h of LPS stimulation and incubation with the different chalcones. The detection has been made by flow cytometry. Dots define single-donor values and the triangles define the mean values. P-values are placed at the lowest value statistically and significantly different from the stimulated control. (b) Plots showing the level of mean TNF-a expression after 6 h of LPS stimulation and incubation with the different chalcones. The detection has been made by flow cytometry. Dots define single-donor values and the triangles define the mean values. P-values are placed at the lowest value statistically and significantly different from the stimulated control.
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inhibition were significant, comparisons between the different groups and the control were carried out by a Tukey’s test or Dunn’s method.
3. Results 3.1. Inhibition of PHA-induced lymphocyte proliferation The inhibitory effects of Licochalcone A (LicA) and five chalcone analogues on phytohemaglutinin (PHA)-induced proliferation were measured by detec-
tion of [3H]-thymidine incorporation. LicA and three of the analogues (PH141, PH100 and PH138) significantly showed dose-dependent inhibitory activity on the proliferation of the lymphocytes (Fig. 1). In contrast, PH105 had no influence on the PHA-induced lymphocyte proliferation (Fig. 1). To further investigate the effect of LicA on lymphocyte proliferation, PKH26 staining and analysis by flow cytometry were performed. Here, LicA also showed a concentration-dependent anti-proliferative effect with lower percentage of lymphocytes in the different offspring generations. Furthermore, the proliferation index was reduced from 1.935 F 0.917
Fig. 5. Plots showing the influence of 25 mg/ml of the chalcones on the TNF-a and IFN-g production in T cells. The detection has been made by flow cytometry and dots define the single-donor values whereas triangles define mean values. (a) Expression of TNF-a. (b) Expression of IFN-g. P-values are placed at mean values statistically and significantly different from the stimulated control.
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(stimulated control) to 1.07 F 0.053 (25 mg/ml LicA) (Fig. 2). 3.2. Inhibition of cytokine secretion by LPS- or PMAstimulated PBMC The inhibitory impact of the chalcones on maximum secretion of TNF-a, IL-1b, IL-6 and IL-10 by LPSstimulated human PBMC was determined following LPS stimulation. PH141, PH100, PH138 and LicA significantly reduced the release of all four cytokines in a concentration-dependent manner (Fig. 3). To investigate the effect of chalcones on IFN-g secretion, PBMCs were stimulated with PMA and ionomycin in the presence of chalcones. IFN-g was significantly reduced by all five chalcones (Fig. 3). 3.3. Inhibition of intracellular cytokines To investigate the inhibitory mechanism of chalcones on cytokine production, intracellular TNF-a and IFN-g levels were detected in T cells and monocytes following stimulation with PMA or LPS. The frequency of TNF-a-producing monocytes after LPS stimulation was significantly reduced by LicA, PH100 and PH138 in a concentration-dependent manner, whereas PH141 and PH105 had no significant effect in concentrations from 0 to 25 mg/ml (Fig. 4a). In contrast, the mean level of TNF-a fluorescence was significantly reduced in a dose-dependent manner by all five chalcones (Fig. 4b). None of the chalcones had an inhibitory effect on the production of TNF-a in T cell, neither in the percentage of TNF-a-producing cells nor in the mean fluorescence of these cells (Fig. 5a). In contrast to TNF-a, the percentage of IFN-g-producing cells was significantly reduced by PH100. The mean level of IFN-g fluorescence was reduced by PH141, PH100 and PH138 (Fig. 5b).
4. Discussion Licochalcone A (LicA), an oxygenated chalcone, inhibits the growth of protozoan parasites and some bacteria both in vitro and in vivo [3,5,6,14 – 17]. It is thus a potential candidate for the treatment of a number of infectious diseases. However, little is known about
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the effects of LicA or its analogues on the cells of the human immune system. In this study, we investigated the effects of LicA and four synthetic analogues (PH141, PH100, PH138 and PH105) on the proliferation ability and cytokine production by human peripheral blood cells. All the analogues in this study had the same chalcone skeleton, but differed in their side chains. We found that LicA and three of the four synthetic analogues (PH141, PH100 and PH138) showed inhibitory activity against PHA-induced human lymphocyte proliferation. In contrast, PH105 did not show an effect on lymphocyte proliferation at the concentrations tested. The fact that only four of the five chalcones had influence on proliferation and that the IC50 values (not shown) differed between the four chalcones indicates that the side chains on the chalcone skeleton are important for the activity of the chalcones. To elaborate on the findings with thymidine, the effect of LicA on proliferation was further investigated by flow cytometry. The flow cytometry data confirmed that LicA has an inhibitory effect on proliferation and indicated that inhibition of proliferation was general phenomena not restricted to any particular T-lymphocyte subsets. Since anti-parasitic drugs such as chloroquine are known to have an effect on cytokine production [9– 11], we investigated the effect of the chalcones on the cytokine production from stimulated leukocytes. To induce the production of the pro-inflammatory cytokines TNF-a, IL-1 and IL-6 and the anti-inflammatory cytokine IL-10, cells were stimulated with LPS, a known stimulator of monocytes through CD14 and toll-like receptors [18 – 22]. PH141, PH100, PH138 and LicA reduced the secretion of both pro- and antiinflammatory cytokines in a dose-dependent manner, indicating a general down regulatory effect of these chalcones on the cytokine production by monocytes. The effect of these chalcones on TNF-a production was further analysed by intracellular detection. These results indicate that the chalcones interfere with the production and not the release of TNF-a. To investigate the effect of Licochalcone A and some of the analogues on cell cytokine production, we stimulated PBMC with PMA and ionomycin, known to induce production of IFN-g from T cells [23]. Again, PH141, PH100, PH138 and LicA inhibited the production of the cytokine in a concentration-dependent
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manner. Not only the release but also the intracellular levels of the cytokine were affected. In contrast to IFN-g, the frequency of T cells producing TNF-a and the levels of TNF-a production were not significantly reduced by any of the tested chalcones following PMA stimulation. However, the production of TNF-a following PMA and ionomycin stimulation was very low, causing the fluorescence in all samples to be close to the background levels. PH105 differed from the rest of the chalcones in regard to cytokine production since it only inhibited the production of IL-1b and IL-10. Taken together with the proliferation data, this suggests less efficient or different mode of action compared to the rest of the chalcones. The present study demonstrates that some chalcones have immunosuppressive activities, which may be taken into consideration when using these for the treatment of infectious diseases. Moreover, these findings indicate that chalcones could be developed into drugs for the treatment of some autoimmune diseases or graft-related symptoms. Acknowledgements This work was supported by grants from the Danish International Development Agency, the Danish Biotechnology Program and the Novo Nordisk Foundation. G. Grauert is thanked for excellent technical support.
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Fich K, et al. Licochalcone A, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of Leishmania. Antimicrob Agents Chemother 1993;37:2550 – 6. Chen M, Christensen SB, Theander TG, Kharazmi A. Antileishmanial activity of licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani. Antimicrob Agents Chemother 1994;38:1339 – 44. Nielsen SF, Christensen SB, Cruciani G, Kharazmi A, Liljefors T. Antileishmanial chalcones: statistical design, synthesis, and three-dimensional quantitative structure – activity relationship analysis. J Med Chem 1998;41:4819 – 32. Nielsen SF, Kharazmi A, Christensen SB. Modifications of the alpha, beta-double bond in chalcones only marginally affect the antiprotozoal activities. Bioorg Med Chem 1998;6:937 – 45. Karres I, Kremer JP, Dietl I, Steckholzer U, Jochum M, Ertel W. Chloroquine inhibits proinflammatory cytokine release into human whole blood. Am J Physiol 1998;274:R1058 – 64. Weber SM, Levitz SM. Chloroquine interferes with lipopolysaccharide-induced TNF-alpha gene expression by a nonlysosomotropic mechanism. J Immunol 2000;165:1534 – 40. Weber SM, Levitz SM. Chloroquine antagonizes the proinflammatory cytokine response to opportunistic fungi by alkalizing the fungal phagolysosome. J Infect Dis 2001;183:935 – 42. Hviid L, Albeck G, Hansen B, Theander TG, Talbot A. A new portable device for automatic controlled-gradient cryopreservation of blood mononuclear cells. J Immunol Methods 1993;157:135 – 42. Kemp K, Bruunsgaard H. Identification of IFN-g producing CD4+ T cells following PMA stimulation. J Interferon Cytokine Res 2001;21:503 – 6. Chen M, Brogger CS, Zhai L, Rasmussen MH, Theander TG, Frokjaer S, et al. The novel oxygenated chalcone, 2,4-dimethoxy-4V-butoxychalcone, exhibits potent activity against human malaria parasite Plasmodium falciparum in vitro and rodent parasites Plasmodium berghei and Plasmodium yoelii in vivo. J Infect Dis 1997;176:1327 – 33. Zhai L, Blom J, Chen M, Christensen SB, Kharazmi A. The antileishmanial agent licochalcone A interferes with the function of parasite mitochondria. Antimicrob Agents Chemother 1995;39:2742 – 8. Zhai L, Chen M, Blom J, Theander TG, Christensen SB, Kharazmi A. The antileishmanial activity of novel oxygenated chalcones and their mechanism of action. J Antimicrob Chemother 1999;43:793 – 803. Hatano T, Shintani Y, Aga Y, Shiota S, Tsuchiya T, Yoshida T. Phenolic constituents of licorice. VIII. Structures of glicophenone and glicoisoflavanone, and effects of licorice phenolics on methicillin-resistant Staphylococcus aureus. Chem Pharm Bull (Tokyo) 2000;48:1286 – 92. Yang RB, Mark MR, Gray A, Huang A, Xie MH, Zhang M, et al. Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling. Nature 1998;395:284 – 8. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 1999;274:10689 – 92. Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, et
L. Barfod et al. / International Immunopharmacology 2 (2002) 545–555 al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998;282:2085 – 8. [21] Kirschning CJ, Wesche H, Merrill AT, Rothe M. Human tolllike receptor 2 confers responsiveness to bacterial lipopolysaccharide. J Exp Med 1998;188:2091 – 7.
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[22] Frey EA, Miller DS, Jahr TG, Sundan A, Bazil V, Espevik T, et al. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med 1992;176:1665 – 71. [23] Paul, WE. Fundamental immunology. Lippincott-Raven, Philadelphia; 1999.
Chalcones from Chinese liquorice inhibit proliferation of T cells and production of cytokines Lea Barfod a, Ka˚re Kemp b,*, Majbritt Hansen a, Arsalan Kharazmi a a Department of Clinical Microbiology, Centre for Medical Parasitology, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark b Department of Infectious Diseases, Centre for Medical Parasitology, M7641, Copenhagen University Hospital (Rigshospitalet), Tagensvej 20, 2200 Copenhagen N, Denmark
Received 20 August 2001; accepted 2 December 2001
Abstract Licochalcone A (LicA), an oxygenated chalcone, has been shown to inhibit the growth of both parasites and bacteria. In this study, we investigated the effect of LicA and four synthetic analogues on the activity of human peripheral blood mononuclear cell proliferation and cytokine production. Four out of five chalcones tested inhibited the proliferation of lymphocytes measured by thymidine incorporation and by flow cytometry. The production of pro- and anti-inflammatory cytokines from monocytes and T cells was also inhibited by four of five chalcones. Furthermore, intracellular detection of cytokines revealed that the chalcones inhibited the production rather than the release of the cytokines. Taken together, these results indicate that LicA and some analogues may have immunomodulatory effects, and may thus be candidates not only as anti-microbial agents, but also for the treatment of other types of diseases. D 2002 Published by Elsevier Science B.V. Keywords: Chalcones; T cells; Cytokines
1. Introduction The discovery of many new drugs against various diseases has been inspired by the use of plants in traditional medicine [1]. Liquorice has been used in China for the treatment of gastric and duodenal ulcers, bronchial asthma, Addison’s disease, food and drug poisoning and skin diseases such as eczema and urticaria [2]. We have reported that Licochalcone A, an oxygenated chalcone originally isolated from Chinese
*
Corresponding author. Tel.: +45-35-45-73-75; fax: +45-3545-76-44. E-mail address: [email protected] (K. Kemp).
liquorice roots, inhibits the in vitro growth of both chloroquine-susceptible and chloroquine-resistant Plasmodium falciparum and protects mice from lethal P. yoelii infections [3]. Others have supported these findings showing inhibitory effects of chalcone analogues on in vitro growth of P. falciparum [4]. Licochalcone A also inhibits the in vitro growth of Leishmania major and L. donovani promastigotes and amastigotes [5], prevents lesion development in mice infected with L. major and reduces the parasite load in the spleen and liver of hamsters infected with L. donovani [6]. Finally, some synthetic chalcone analogues of LicA have also shown comparable activities to LicA [7,8]. Some anti-protozoan drugs interfere with the immune response. Among others, the most prominent
1567-5769/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 1 5 6 7 - 5 7 6 9 ( 0 1 ) 0 0 2 0 2 - 8
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anti-malaria drug, chloroquine [9 – 11], inhibits the release of pro-inflammatory cytokines [9]. Since LicA and its analogues are potential antiprotozoan drugs, the effect of these on the human immune system is important. To this end, we have investigated the influence of LicA and synthetic analogues on cytokine production and lymphocyte proliferation.
2. Materials and methods 2.1. Chalcones Licochalcone A (LicA) and 21 synthetic analogues were synthesized at the Department of Medicinal Chemistry, Royal Danish School of Pharmacy (Copenhagen, Denmark). LicA and four of the analogues were selected and included in this study based on the effect on LPS-induced TNF-a secretion. Chalcones were diluted in DMSO and RPMI 1640 to a maximum final concentration of 0.5%. 2.2. Isolation of mononuclear cells and viability Heparinized peripheral blood (20 ml) was collected from healthy Danish donors. Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep (Nyegaard, Oslo, Norway) density centrifugation and washed twice in RPMI 1640. Finally, the cells were dissolved in growth media (RPMI 1640 containing 10% human serum, 40 IU/ml penicillin/streptomycin (Gibco, Paisley, UK) and 1.6 mM L -glutamine (Sigma-Aldrich, Germany) or cryopreserved in liquid nitrogen as described previously [12]. Prior to experiments, frozen PBMCs were rapidly thawed, washed and the viability was determined by trypan blue staining. Incubation with 25 mg/ml of LicA or the analogues PH141, PH138, or PH105 resulted in no less than 93% viability. The cells incubated with 25 mg/ml of PH100 had less than 80% viability. At 12.5 mg/ml, this viability was increased to more than 97%. 2.3. Measurement of lymphocyte proliferation by [3H]-thymidine incorporation Frozen PBMCs were rapidly thawed, washed and the viability was determined by trypan blue staining.
Wells of round-bottomed plates received 100 ml of PBMC (1 106 cells/ml) in growth media. PHA (20 ml) at a final concentration of 40 mg/ml and 20 ml of medium were added to test cultures and control cultures, respectively. Chalcone dilutions were added after incubation for 2 days at 37 C in a humidified atmosphere containing 5% CO 2. The cells were pulsed with 20 ml/well (1.85 Mbq/well) of [3H]thymidine (New England Nuclear, Boston, MA, USA) after incubation for another 2 h at 37 C. Cells were harvested onto glass fibre filters after 20 h and the incorporation of [3H]-thymidine into DNA was determined by a Matrix b-counter (Packard, Greve, Denmark). 2.3.1. Cytokine measurements in culture supernatants Frozen PBMCs were rapidly thawed and washed. The cells were stimulated for 22 h at 37 C with either 10 ng/ml E. coli LPS (Difco, Detroit, MI) or PMA (Sigma-Aldrich) and ionomycin (Calbiochem, USA) (final concentrations of 0.1 and 3.5 mg/ml) and with or without dilutions of the chalcones at a final concentration of 0– 100 mg/ml. The concentrations of IFN-g, TNF-a, IL-1b, IL-6 and IL-10 in the supernatants were measured by ELISA in triplicates (R&D Systems, London, England). TNF-a, IL-1b, IL-6 and IL-10 concentrations were measured following LPS stimulation and IFN-g was measured after PMA and ionomycin stimulation. 2.3.2. Flow cytometry 2.3.2.1. Intracellular cytokine detection. For the measurements of intracellular cytokines in T cells, monensin (1.5 mM; Sigma, St. Louis, MO, USA), ionomycin (1 mM), PMA (50 ng/ml) and chalcone dilutions (25 mg/ml) were added to PBMC cultures in polystyrene tubes for 4 h. Measurement of intracellular cytokines in monocytes was done in the following manner: PBMCs were stimulated 6 h in polystyrene tubes at 37 C with LPS (final concentration of 10 ng/ml), monensin (final concentration of 0.001 mg/ml) (Sigma-Aldrich) and 0– 25 mg/ml of the chalcones. The intracellular staining procedure was based on other studies [13]. In brief, following stimulation, cells were harvested and washed in PBS, resuspended in staining buffer (PBS, 0.5% BSA, 0.01% NaN3) and
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labelled with antibodies directed against the T-cell surface marker CD3 (Dako, Glostrup, Denmark) for 20 min at room temperature. The cells were washed twice in staining buffer, fixed in staining buffer containing 2% formaldehyde (Sigma) and then washed twice in staining buffer and twice in freshly made saponin buffer (staining buffer containing 0.1% (w/v) saponin (Sigma)). Finally, cells were incubated with anti-cytokine (IFN-g or TNF-a, Pharmingen, CA, USA) antibodies for 30 min. Following cytokine labelling, the cells were washed twice in saponin buffer, twice in staining buffer, resuspended and analysed on an EpicsXL-MCL flow-cytometer (Coulter, Miami, FL, USA). Flow cytometric data were analysed by Winlist 3D software (Verity Software House, ME, USA). Samples were live-gated on lymphocytes or monocytes by forward and side scatter gates and limits for fluorescence positive cells were set using nonspecific IgG antibodies.
colour reaction was terminated by the addition of heat-inactivated fetal calf serum (HFCS). The cell suspension was diluted in RPMI 1640 supplemented with 58.4 mg/ml L-glutamine, 20 IU/ml penicillin/ streptomycin (Gibco) and 10% HFCS and washed twice. Finally, the cells were resuspended in growth media (seen previously) at a cell density of 2 106/ ml. The cells were incubated with or without (DMSO alone) 125-ml chalcone dilutions and with or without 50-ml PHA for 3 days. Following incubation, cells were incubated 20 min in formalin buffer and washed twice in PBS supplemented with 10% HFCS. Flow cytometric analyses were performed using Modfit LT 3.0 software (Verity Software House). The unstimulated control samples were used to determine the parent peak and, on this basis, the next generations were defined as the cells having proliferated one to three times. The proliferation index was calculated according to the Modfit LT 3.0 software.
2.3.3. Lymphocyte proliferation Freshly isolated PBMCs were washed twice in PBS and after the last wash, pellet was dissolved in Diluent C (Sigma-Aldrich) and rapidly added the fluorescent membrane due PKH26 solution (1:250 in Diluent C) (Sigma-Aldrich). After 3 min at RT, the
2.4. Statistical analysis All statistical calculations were performed using Sigma Stat software 2.0 (SPSS Science, USA). Initial analyses were one-way ANOVA or Kruskal – Wallis one-way ANOVA on ranks. If the P-values for the
Fig. 1. Plot showing the inhibition of PHA-stimulated lymphocytes by five chalcones. The concentration of DMSO in the control is similar to the concentrations in the chalcone dilutions. The values are mean of three independent triple determinations and error bars indicate the standard deviation from the mean.
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Fig. 2. (a) Representative flow cytometry data showing the PKH26 fluorescence of lymphocytes and the separation of the different populations. Left: the unstimulated control defining the parent peak; middle: PHA-stimulated control; right: LicA and PHA-stimulated lymphocytes. (b) Histogram showing the percentage of lymphocytes in different generations determined by PKH26 staining. The numbers in top of the columns are the proliferation indexes. The values are mean values from four independent determinations with different donors. Standard deviations from the mean are shown by error bars. Asterisks indicate proliferation indexes statistically and significantly lower compared to the stimulated control. The concentration of DMSO in the DMSO control is 0.25%, which is the same as in the highest LicA dilution (25 mg/ml).
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Fig. 3. Graphs showing the inhibition of cytokine secretion from LPS-stimulated (A – D) or PMA- and ionomycin-stimulated (E) PBMC by five chalcones. Values are mean of three to six independent triple determinations with different donors. Error bars define the standard deviation.
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Fig. 4. (a) Plots showing the frequency of monocytes expressing TNF-a after 6 h of LPS stimulation and incubation with the different chalcones. The detection has been made by flow cytometry. Dots define single-donor values and the triangles define the mean values. P-values are placed at the lowest value statistically and significantly different from the stimulated control. (b) Plots showing the level of mean TNF-a expression after 6 h of LPS stimulation and incubation with the different chalcones. The detection has been made by flow cytometry. Dots define single-donor values and the triangles define the mean values. P-values are placed at the lowest value statistically and significantly different from the stimulated control.
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inhibition were significant, comparisons between the different groups and the control were carried out by a Tukey’s test or Dunn’s method.
3. Results 3.1. Inhibition of PHA-induced lymphocyte proliferation The inhibitory effects of Licochalcone A (LicA) and five chalcone analogues on phytohemaglutinin (PHA)-induced proliferation were measured by detec-
tion of [3H]-thymidine incorporation. LicA and three of the analogues (PH141, PH100 and PH138) significantly showed dose-dependent inhibitory activity on the proliferation of the lymphocytes (Fig. 1). In contrast, PH105 had no influence on the PHA-induced lymphocyte proliferation (Fig. 1). To further investigate the effect of LicA on lymphocyte proliferation, PKH26 staining and analysis by flow cytometry were performed. Here, LicA also showed a concentration-dependent anti-proliferative effect with lower percentage of lymphocytes in the different offspring generations. Furthermore, the proliferation index was reduced from 1.935 F 0.917
Fig. 5. Plots showing the influence of 25 mg/ml of the chalcones on the TNF-a and IFN-g production in T cells. The detection has been made by flow cytometry and dots define the single-donor values whereas triangles define mean values. (a) Expression of TNF-a. (b) Expression of IFN-g. P-values are placed at mean values statistically and significantly different from the stimulated control.
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(stimulated control) to 1.07 F 0.053 (25 mg/ml LicA) (Fig. 2). 3.2. Inhibition of cytokine secretion by LPS- or PMAstimulated PBMC The inhibitory impact of the chalcones on maximum secretion of TNF-a, IL-1b, IL-6 and IL-10 by LPSstimulated human PBMC was determined following LPS stimulation. PH141, PH100, PH138 and LicA significantly reduced the release of all four cytokines in a concentration-dependent manner (Fig. 3). To investigate the effect of chalcones on IFN-g secretion, PBMCs were stimulated with PMA and ionomycin in the presence of chalcones. IFN-g was significantly reduced by all five chalcones (Fig. 3). 3.3. Inhibition of intracellular cytokines To investigate the inhibitory mechanism of chalcones on cytokine production, intracellular TNF-a and IFN-g levels were detected in T cells and monocytes following stimulation with PMA or LPS. The frequency of TNF-a-producing monocytes after LPS stimulation was significantly reduced by LicA, PH100 and PH138 in a concentration-dependent manner, whereas PH141 and PH105 had no significant effect in concentrations from 0 to 25 mg/ml (Fig. 4a). In contrast, the mean level of TNF-a fluorescence was significantly reduced in a dose-dependent manner by all five chalcones (Fig. 4b). None of the chalcones had an inhibitory effect on the production of TNF-a in T cell, neither in the percentage of TNF-a-producing cells nor in the mean fluorescence of these cells (Fig. 5a). In contrast to TNF-a, the percentage of IFN-g-producing cells was significantly reduced by PH100. The mean level of IFN-g fluorescence was reduced by PH141, PH100 and PH138 (Fig. 5b).
4. Discussion Licochalcone A (LicA), an oxygenated chalcone, inhibits the growth of protozoan parasites and some bacteria both in vitro and in vivo [3,5,6,14 – 17]. It is thus a potential candidate for the treatment of a number of infectious diseases. However, little is known about
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the effects of LicA or its analogues on the cells of the human immune system. In this study, we investigated the effects of LicA and four synthetic analogues (PH141, PH100, PH138 and PH105) on the proliferation ability and cytokine production by human peripheral blood cells. All the analogues in this study had the same chalcone skeleton, but differed in their side chains. We found that LicA and three of the four synthetic analogues (PH141, PH100 and PH138) showed inhibitory activity against PHA-induced human lymphocyte proliferation. In contrast, PH105 did not show an effect on lymphocyte proliferation at the concentrations tested. The fact that only four of the five chalcones had influence on proliferation and that the IC50 values (not shown) differed between the four chalcones indicates that the side chains on the chalcone skeleton are important for the activity of the chalcones. To elaborate on the findings with thymidine, the effect of LicA on proliferation was further investigated by flow cytometry. The flow cytometry data confirmed that LicA has an inhibitory effect on proliferation and indicated that inhibition of proliferation was general phenomena not restricted to any particular T-lymphocyte subsets. Since anti-parasitic drugs such as chloroquine are known to have an effect on cytokine production [9– 11], we investigated the effect of the chalcones on the cytokine production from stimulated leukocytes. To induce the production of the pro-inflammatory cytokines TNF-a, IL-1 and IL-6 and the anti-inflammatory cytokine IL-10, cells were stimulated with LPS, a known stimulator of monocytes through CD14 and toll-like receptors [18 – 22]. PH141, PH100, PH138 and LicA reduced the secretion of both pro- and antiinflammatory cytokines in a dose-dependent manner, indicating a general down regulatory effect of these chalcones on the cytokine production by monocytes. The effect of these chalcones on TNF-a production was further analysed by intracellular detection. These results indicate that the chalcones interfere with the production and not the release of TNF-a. To investigate the effect of Licochalcone A and some of the analogues on cell cytokine production, we stimulated PBMC with PMA and ionomycin, known to induce production of IFN-g from T cells [23]. Again, PH141, PH100, PH138 and LicA inhibited the production of the cytokine in a concentration-dependent
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manner. Not only the release but also the intracellular levels of the cytokine were affected. In contrast to IFN-g, the frequency of T cells producing TNF-a and the levels of TNF-a production were not significantly reduced by any of the tested chalcones following PMA stimulation. However, the production of TNF-a following PMA and ionomycin stimulation was very low, causing the fluorescence in all samples to be close to the background levels. PH105 differed from the rest of the chalcones in regard to cytokine production since it only inhibited the production of IL-1b and IL-10. Taken together with the proliferation data, this suggests less efficient or different mode of action compared to the rest of the chalcones. The present study demonstrates that some chalcones have immunosuppressive activities, which may be taken into consideration when using these for the treatment of infectious diseases. Moreover, these findings indicate that chalcones could be developed into drugs for the treatment of some autoimmune diseases or graft-related symptoms. Acknowledgements This work was supported by grants from the Danish International Development Agency, the Danish Biotechnology Program and the Novo Nordisk Foundation. G. Grauert is thanked for excellent technical support.
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