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Effect of a garlic derivative (alliin) on peripheral blood cell immune responses
International Journal of Immunopharmacology 21 (1999) 589±597
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Eect of a garlic derivative (alliin) on peripheral blood cell immune responses Hertzel Salman a,b, Michael Bergman a,b, Hanna Bessler c, Igor Punsky c, Meir Djaldetti c,* a
Department of Medicine `C', Rabin Medical Center, Golda Campus, 7 Keren Kayemet Street, Petah Tiqva, Israel b The Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel c The Laboratory for Immunology and Hematology Research, Rabin Medical Center, Golda Campus, 7 Keren Kayemet Street, Petah Tiqva, Israel Received 1 February 1999; accepted 6 May 1999
Abstract The in vitro eect of the garlic derivative alliin, on the mitogen induced peripheral blood mononuclear cell (PBMC) proliferation and cytokine production was examined. In addition, its eect on the engul®ng capacity of phagocyting cells was evaluated. The results showed an increase in pokeweed mitogen (PWM) induced cell proliferation, IL-1b and TNF-a production, as well as in the engul®ng capacity of both percentage of phagocyting cells and number of latex particles phagocytized by each individual cell. The Con-A induced cell proliferation and IL-6 production decreased following incubation with alliin, whereas PHA-induced cell proliferation, IL-2 and superoxide anion generation remained unchanged. It is concluded that alliin in vitro exerts an immunomodulatory eect on certain functions of the peripheral blood cells. # 1999 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Garlic; Alliin; Cytokines; Phagocytosis
1. Introduction Garlic is a plant-based phytomedicine with a long history of therapeutic activity against parasites, worms, bacterial and fungal infections [5] and even with anti-cancer [15,1] and anti* Corresponding author. Tel.: +1-972-3-9372398; fax: +1-972-3-9372332. E-mail address: [email protected] (M. Djaldetti) 0192-0561/99/$20.00 # 1999 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 9 9 ) 0 0 0 3 8 - 7
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H. Salman et al. / International Journal of Immunopharmacology 21 (1999) 589±597
thrombotic [7,8] properties. In addition, there is a wide-spread belief that garlic prolongs life by lowering blood pressure and cholesterol level and consequently Ð heart attacks [14,19,20]. No wonder, therefore, that with an increasing interest in herbal and alternative medicine, the use of garlic and its derivatives is presently an issue with a huge and pro®t bearing market. Conversely, well-controlled studies showed that garlic in its various forms was ineective in lowering either serum cholesterol level [5], or elevated blood pressure [25]. As for the preventive eect of garlic on infections, the opinions about its ecacy as a drug and its mechanism of action vary in dierent publications. Most studies underline the anti-oxidative eect of garlic and its derivatives as the principal factor for its anti-microbial properties [3,18,22]. On the other hand, little is known about the interrelationship of garlic and the immune system. Therefore, it was the aim of the present study to examine the in vitro eect of the garlic derivative alliin on cytokine production by peripheral blood mononuclear cells (PBMC) from healthy individuals and their mitogen response. In addition, its ability to aect the superoxide anion production and phagocytic function of peripheral blood leukocytes and monocytes was tested.
2. Materials and methods 2.1. Alliin The allicin precursor alliin (Inodail capsules containing 4000 mg alliin each), was kindly supplied by Pharma Guri LTD, Israel. 2.2. Cells Peripheral blood mononuclear (PBMC) and polymorphonuclear (PMN) cells were isolated from heparinized venous blood of 25 healthy volunteers using a Ficoll Paque gradient centrifugation [4]. The cells were suspended in RPMI-1640 medium containing 1% penicillin, streptomycin and nystatin, and supplemented with 5% fetal calf serum (FCS-M) (Biological Industries, Beit Haemek, Israel). 2.3. Cytokine production PBMC (2 106) suspended in 1 ml FCS-M were incubated without or with 10 ng/ml lipopolysaccharide (Escherichia coli, LPS, Sigma) for spontaneous and LPS induced secretion of IL-1b, with LPS for IL-6 and TNFa production and with 1% phytohaemagglutinin (PHAM, Difco) for IL-2 production. Alliin was added at the onset of the cultures at the following concentrations: 0.05; 1.0; and 3.0 mg/ml. These doses were calculated on a body weight basis extrapolated from the therapeutic dosage recommended by the company. Cells incubated in the absence of alliin served as controls. Culture media were collected at 24 h (for IL-1b, IL-6 and TNFa), or at 48 h for IL-2, the cells were removed by centrifugation and the supernatants were kept at ÿ708C until assayed. Cytokine concentration in the supernatants was tested using ELISA kits speci®cally for
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human IL-1b, IL-6, TNFa (Genzyme Corporation, Cambridge, MA) and IL-2 (R&D systems, Minneapolis, MN, USA) as detailed in the guide-line provided by the manufacturer. The detection levels of these cytokines in the assays were 30 pg/ml for IL-1b, IL-2 and TNFa and 15 pg/ml for IL-6. 2.4. Mitogen response Aliquots of PBMC (0.1 ml) suspension (2 106 ml) were divided into each well of 96 well plates (¯at bottom, Nunc) containing 0.1 ml of CM or 2% phytohemagglutinin (PHA-M, Difco), 10 mg/ml concanavalin A (Con A), or 20 mg/ml pokeweed mitogen (PWM, Sigma). Alliin was added at the onset of the culture at concentrations ranging between 0.5 and 3.0 mg/ ml. Cultures incubated in the absence of alliin served as controls. Triplicates were incubated for 3 d. Eighteen hours before harvesting, 3H-TdR (Thymidine-methyl-3H,4 Ci/mmole, ICN), 0.5 mCi/well, was added to the culture media. Radioactivity was measured with a LKB liquid scintillation counter model 3380. 2.5. Superoxide anion production Superoxide anion generation by PBMC and PMN cells was examined by the method of Johnston et al. [6]. Brie¯y, 4 106 cells suspended in 1 ml of PBS supplemented with 1 gr% glucose, 0.5 mM CaCl2 and 80 nM cytochrome C (from horse heart, Sigma, St. Louis, MD, USA) were incubated at 378C for 60 min without or with 2 10ÿ3 mM phorbol meristate acetate (PMA, Sigma) and without and with 3.0 mg/ml of alliin. After centrifugation at 1500 g, the supernatants were collected and the absorbency was determined at 550 nm. The amount of superoxide anions produced was directly proportional to the amount of reduced cytochrome C, calculated by subtracting the OD values without PMA, from those obtained with PMA. The speci®city of the reaction was con®rmed by complete inhibition of cytochrome C reduction following the addition of superoxide dismutase to the incubation mixture. 2.6. Phagocytosis Uniform polystyrene latex particles (0.1 ml of 5% suspension) (0.8 mm in diameter, Difco, Detroit, MI) in PBS were added to 106 ml of PMN cells incubated without and with 3.0 mg/ml of alliin. After 60 min the cells were sedimented, ®xed in cold 1% glutaraldehyde in phosphate buer pH 7.4, post®xed with osmium tetroxide and embedded in agar-100 resin (Agar Scienti®c LTD Cambridge, UK). Semi-thick sections cut with a LKB ultratome III were stained with methylene blue and the cells which engulfed latex particles, as well as the number of particles phagocytized by each individual cell were counted using a light microscope. At least 200 cells from each individual were examined. 2.7. Statistical evaluation Statistical analysis was carried out using repeated measures and paired t-test within each group. Results are expressed as mean2 SEM.
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3. Results
3.1. Mitogen induced proliferation (Fig 1) Concentrations of alliin between 0.5 and 3.0 mg/ml signi®cantly reduced the proliferative response of PBMC to Con A in a dose dependent manner down to 30% for the maximal concentration used. Alliin at concentrations of 1 and 3.0 mg/ml stimulated cell proliferation induced by PWM by 39 and 45%, respectively. Alliin had no eect on PHA-induced cell proliferation.
3.2. Cytokine production Incubation of PBMC with alliin at the three concentrations used in the study induced a signi®cant increase in spontaneous IL-1b production up to 30% when 3.0 mg/ml were applied. In LPS-treated cells an increase was observed only with the highest concentration used (14%, Fig. 2). Alliin did not have any eect on IL-2 production. As for its eect on IL-6, only the highest concentration used in the experiments caused a signi®cant decrease in both spontaneous and LPS-induced production by 22 and 30%, respectively (Fig. 3). There was no dierence in TNFa generation by LPS stimulated and non-stimulated cells incubated with 1 mg/ ml of alliin.
Fig. 1. Eect of various concentrations of alliin on mitogen induced cell proliferation. p < 0.04
p < 0.01.
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Fig. 2. Eect of dierent concentrations of alliin on spontaneous and LPS-induced IL-1b production. p < 0.05 p < 0.01 p < 0.001.
3.3. Oxygen superoxide production Alliin, at all three concentrations applied in the study did not exert any signi®cant eect on by both PMN and PBMC, the values being, 10.320.8 and 7.9 20.8 nm/106 cells, respectively, without alliin, vs 9.42 0.6 and 7.4 20.9 nm/106 following incubation with 3.0 mg/ml of alliin. Oÿ 2 -production
Fig. 3. Eect of alliin on spontaneous and LPS-induced IL-6 production. p < 0.05
p < 0.001.
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3.4. Phagocytosis Incubation of PMN cells with alliin caused a signi®cant increase in both the percentage of phagocyting cells, from 48.322.8 to 56.821.8% ( p < 0.02) and the number of latex particles engulfed by each individual cell, from 9.2 20.8 to 14.521.0 particles/cell ( p < 0.002, Fig. 4).
4. Discussion For many years, garlic has been recognized as a valuable folk medicine with a favorable eect against a large number of pathological conditions [2,23]. Powdered material obtained from garlic bulbs contains about 1% of alliin, which in the presence of the enzyme alliinase converts to allicin. It is accepted that the bene®cial properties of the garlic are due to allicin [22] and its transformation products which include ajoenes, vinyldithines, and polysul®des. Lachmann et al. [11] compared the pharmacokinetics of S35 labeled alliin, allicin and vinyldithines orally administered to rats and found that the blood activity pro®le for alliin diered considerably from that for allicin and vinyldithines. In their studies the maximum blood level for alliin was reached within 10 min and eliminated almost completely from the blood after 6 h. As for the remaining two garlic constituents, the maximum blood values were 30±60 min for allicin and 120 min for vinyldithines and values of more than 1000 ng-Eq/ml persisted at the end of the study after 72 h. While both allicin and alliin were shown to possess signi®cant antioxidant activity, the biological eect of allicin is attributed to its rapid reaction with thiol containing proteins [22], whereas that of alliin is due to its capacity to scavenge hydroxyl radicals [10,21].
Fig. 4. Percent of phagocyting cells and number of latex particles engulfed by each individual cell without and with alliin. p < 0.02 p < 0.002.
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The long list of conditions which may be prevented and treated with garlic and its compounds has brought about the widespread belief that garlic possesses a great therapeutic potential including immunostimulation [2,23]. The results of the present study showed that alliin in vitro exerts a certain eect on the immune response of peripheral blood cells. This eect consisted of an increase in PWM induced cell proliferation, spontaneous production of IL-1b, as well as an increase in both number of phagocyting cells and latex particles engulfed by each individual cell. Alliin caused a decrease in Con A induced cell proliferation and IL-6 production, but did not have any eect on PHA induced cell proliferation, IL-2 and superoxide anion generation. Since PWM is known to be a B-cell stimulator and an inducer of immunoglobulin synthesis, the increased proliferative response to PWM suggests an enhancing eect of alliin on B-lymphocytes for antibody production. The decreased response to Con-A, an activator of a subset of suppressor cells, indicates a possible immunomodulatory eect of alliin via reduction of suppressor cells. T cells activated with Con A may either suppress or stimulate B cells to proliferate or dierentiate to plasma cells [13]. Using anti-Tac, a speci®c monoclonal antibody, Con A induced stimulation of two types of human T cells, i.e. Tac (+) with and Tac (ÿ) without suppressive activity [27,28]. Moreover, mitogen stimulation of T cells was shown to be associated with membranal changes in various cell phenotypes resulting in an alteration of their susceptibility to drugs [9,16]. Therefore, the possibility that alliin exerts dierent eects on various cell populations comprising the PBMC cannot be excluded. Following activation with PHA, certain populations of resting T cells enter cell cycle and divide rapidly to produce large numbers of progenitors that will dierentiate into armed eector T cells. IL-2 produced by activated T cells drives T cell proliferation and dierentiation. The present ®ndings indicate that alliin aects speci®c lymphocyte subsets, but not T cells responding to PHA. The observed alteration in IL-1b and IL-6 production, which are known as proin¯ammatory cytokines, suggest an eect of alliin on the in¯ammatory reactions. Previous reports con®rm the role of garlic derivatives as immunomodulators. Morioka et al. [17] have reported that F4, a protein fraction isolated from aged garlic extract enhanced the cytotoxicity of human lymphocytes against natural killer (NK) sensitive K562 and NK-resistant M14 cell lines. This eect further increased when F4 was administered together with suboptimal doses of IL-2, a ®nding suggesting the existence of an enhancing eect of IL-2 on F4. An augmentation of Tlymphocyte blastogenesis by garlic compounds was reported by Lau et al. [12] indicating that garlic possesses a bene®cial immuno-stimulatory function. Romano et al. [24] have shown that ajoene, a compound isolated from garlic ethanolic extracts exerted a strong inhibitory eect on PHA, phorbol myristate and anti-CD3 induced proliferation of human lymphocytes, as well as LPS-induced TNF generation. Although our results did not show any eect of alliin on PHA induced proliferation, the stimulation of lymphocyte proliferation induced by PWM and the inhibition caused by Con A con®rm the eect of garlic extracts on lymphocyte membranedependent functions. The dierence between Romano's et al. [24] and our ®ndings might be due to the dierent forms of garlic preparations used in the studies. The stimulation of the phagocytic activity in the present study is of interest and further con®rms the value of garlic preparation for disease prevention. Increased phagocytic and immune functions induced by garlic preparation has been reported also by Sumiyoshi [26].
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In summary, the present results indicate that the garlic derivative alliin possess, at least in vitro, a stimulatory eect on certain peripheral blood cell immune functions, as well as on the engul®ng capacity of phagocytic cells. Acknowledgements This work was supported by the generous help provided by Mr Ophir Perlson, Atmor, BneiBrak, Israel. References [1] Agarwal KC. Therapeutic actions of garlic constituents. Med Res Rev 1996;16:111±24. [2] Augusti KT. Therapeutic values of onion (Allium cepa L.) and garlic (Allium sativum L.). Indian J Exp Biol 1996;34:634±40. [3] Geng Z, Rong Y, Lau BH. S-allyl cysteine inhibits activation of nuclear factor kappa B in human T cells. Free Radic Biol Med 1997;23:345±50. [4] Hellstrand K, Hermodsson S. Interleukin-2 can induce suppression of human natural killer cell cytotoxicity. Clin Exp Immunol 1989;7:410±6. [5] Isaacsohn JL, Moser M, Stein EA, Dudley K, Davey JA, Liskov E, Black HR. Garlic powder and plasma lipids and lipoproteins. A multicenter, randomized, placebo-controlled trial. Arch Int Med 1998;158:1189±94. [6] Johnston RB, Godzik CA, Cohn ZA. Increased superoxide anion production by immunologically activated and chemically elicited macrophages. J Exp Med 1978;148:115±27. [7] Kiesewetter H, Jung F, Jung EM, Mroweitz C, Koscielny J, Wenzel E. Eect of garlic on platelet aggregation in patients with increased risk of juvenile ischemic attack. Eur J Clin Pharmacol 1993;45:333±6. [8] Kiesewetter H, Jung F, Pindur G, Jung EM, Mroweitz C, Wenzel E. Eect of garlic on thrombocyte aggregation, microcirculation, and other risk factors. Int J Clin Pharmacol Ther Toxicol 1991;29:151±5. [9] Komlos L, Ben-Efraim S, Notmann J, Hart J, Halbrecht I. Dierence in sensitivity to melphalan between ConA-activated and nonactivated human T-cell subsets. Clin Immunol Immunopathol 1987;45:105±13. [10] Kourounakis PN, Rekka EA. Eect of active oxygen species of alliin and Allium sativum (garlic) powder. Res Commun Chem Pathol, Pharmacol 1991;74:249±52. [11] Lachmann G, Lorenz D, Radeck W, Steiper M. The pharmacokinetics of the S35 labeled garlic constituents alliin, allicin and vinyldithine. Arzneimittelforshung 1994;44:734±43. [12] Lau BH, Yamasaki T, Gridley DC. Garlic compounds modulate macrophage and T-lymphocyte function. Mol Biother 1991;3:103±7. [13] Lydyard PM, Hayward AR. Induction of suppression through human T cell interactions. Clin Exp Immunol 1979;39:496±502. [14] McMahon FG, Vargas R. Can garlic lower blood pressure? A pilot study. Pharmacotherapy 1993;13:406±7. [15] Milner JA. Garlic: its anticarcinogenic and antitumorigenic properties. Nutr Rev 1996;54:S82±S86. [16] Moore K, Nesbitt AM. Identi®cation and isolation of OKT4+suppressor cells with the monoclonal antibody WR16. Immunology 1986;58:659±64. [17] Morioka N, Sze LL, Morton DL, Irie RF. A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin A. Cancer Immunol Immunother 1993;37:316±22. [18] Numagami Y, Sato S, Ohnishi ST. Attenuation of rat ischemic brain damage by aged garlic extracts: a possible protecting mechanism as antioxidants. Neurochem Int 1996;29:135±43. [19] Orekhov AN, Tertov VV, Sobenin IA, Pivovarova EM. Direct anti-atherosclerosis-related eects of garlic. Ann Med 1995;27:63±5. [20] Phelps S, Harris WS. Garlic supplementation and lipoprotein oxidation susceptibility. Lipids 1993;28:475±7.
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[21] Prasad K, Laxdal VA, Yu M, Raney BL. Antioxidant activity of allicin, an active principle in garlic. Mol Cell Biochem 1995;148:183±9. [22] Rabinkov A, Miron T, Konstantinovski L, Wilchek M, Mirelman D, Weiner L. The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins. Biochim Biophys Acta 1998;1379:233±44. [23] Resch KL, Ernst E. Garlic (Allium sativum)-a potent medicinal plant. J Fortschr Med 1995;113:311±5. [24] Romano EL, Montano RF, Brito B, Apitz R, Alonso J, Romano M, Gebran S, Soyano A. Eect of Ajoene on lymphocyte and macrophage membrane-dependent functions. Immunopharmacol Immunotoxicol 1997;19:15± 36. [25] Simons LA, Balasubramaniam S, von-Konigsmark M, Par®tt A, Simons J, Peters W. On the eect of garlic on plasma lipids and lipoprotein in mild hypercholesterolemia. Atherosclerosis 1995;113:219±25. [26] Sumiyoshi H. New pharmacological activities of garlic and its constituents. Nippon Yakurigaku Sasshi 1997;110(1):93±7. [27] Uchiyama T, Broder S, Waldmann TA. Monoclonal antibody (anti Tac) reactive with activated and functionally mature T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J Immunol 1981;126:1393±7. [28] Uchiyama T, Nelson DL, Fleisher TA, Waldmann TA. Monoclonal antibody (anti Tac) reactive with activated and functionally mature T cells. II. Expression of Tac antigen on activated cytotoxic killer T cells, suppressor cells, and one of two types of helper T cells. J Immunol 1981;126:1398±403.
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Eect of a garlic derivative (alliin) on peripheral blood cell immune responses Hertzel Salman a,b, Michael Bergman a,b, Hanna Bessler c, Igor Punsky c, Meir Djaldetti c,* a
Department of Medicine `C', Rabin Medical Center, Golda Campus, 7 Keren Kayemet Street, Petah Tiqva, Israel b The Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel c The Laboratory for Immunology and Hematology Research, Rabin Medical Center, Golda Campus, 7 Keren Kayemet Street, Petah Tiqva, Israel Received 1 February 1999; accepted 6 May 1999
Abstract The in vitro eect of the garlic derivative alliin, on the mitogen induced peripheral blood mononuclear cell (PBMC) proliferation and cytokine production was examined. In addition, its eect on the engul®ng capacity of phagocyting cells was evaluated. The results showed an increase in pokeweed mitogen (PWM) induced cell proliferation, IL-1b and TNF-a production, as well as in the engul®ng capacity of both percentage of phagocyting cells and number of latex particles phagocytized by each individual cell. The Con-A induced cell proliferation and IL-6 production decreased following incubation with alliin, whereas PHA-induced cell proliferation, IL-2 and superoxide anion generation remained unchanged. It is concluded that alliin in vitro exerts an immunomodulatory eect on certain functions of the peripheral blood cells. # 1999 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Garlic; Alliin; Cytokines; Phagocytosis
1. Introduction Garlic is a plant-based phytomedicine with a long history of therapeutic activity against parasites, worms, bacterial and fungal infections [5] and even with anti-cancer [15,1] and anti* Corresponding author. Tel.: +1-972-3-9372398; fax: +1-972-3-9372332. E-mail address: [email protected] (M. Djaldetti) 0192-0561/99/$20.00 # 1999 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 9 9 ) 0 0 0 3 8 - 7
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H. Salman et al. / International Journal of Immunopharmacology 21 (1999) 589±597
thrombotic [7,8] properties. In addition, there is a wide-spread belief that garlic prolongs life by lowering blood pressure and cholesterol level and consequently Ð heart attacks [14,19,20]. No wonder, therefore, that with an increasing interest in herbal and alternative medicine, the use of garlic and its derivatives is presently an issue with a huge and pro®t bearing market. Conversely, well-controlled studies showed that garlic in its various forms was ineective in lowering either serum cholesterol level [5], or elevated blood pressure [25]. As for the preventive eect of garlic on infections, the opinions about its ecacy as a drug and its mechanism of action vary in dierent publications. Most studies underline the anti-oxidative eect of garlic and its derivatives as the principal factor for its anti-microbial properties [3,18,22]. On the other hand, little is known about the interrelationship of garlic and the immune system. Therefore, it was the aim of the present study to examine the in vitro eect of the garlic derivative alliin on cytokine production by peripheral blood mononuclear cells (PBMC) from healthy individuals and their mitogen response. In addition, its ability to aect the superoxide anion production and phagocytic function of peripheral blood leukocytes and monocytes was tested.
2. Materials and methods 2.1. Alliin The allicin precursor alliin (Inodail capsules containing 4000 mg alliin each), was kindly supplied by Pharma Guri LTD, Israel. 2.2. Cells Peripheral blood mononuclear (PBMC) and polymorphonuclear (PMN) cells were isolated from heparinized venous blood of 25 healthy volunteers using a Ficoll Paque gradient centrifugation [4]. The cells were suspended in RPMI-1640 medium containing 1% penicillin, streptomycin and nystatin, and supplemented with 5% fetal calf serum (FCS-M) (Biological Industries, Beit Haemek, Israel). 2.3. Cytokine production PBMC (2 106) suspended in 1 ml FCS-M were incubated without or with 10 ng/ml lipopolysaccharide (Escherichia coli, LPS, Sigma) for spontaneous and LPS induced secretion of IL-1b, with LPS for IL-6 and TNFa production and with 1% phytohaemagglutinin (PHAM, Difco) for IL-2 production. Alliin was added at the onset of the cultures at the following concentrations: 0.05; 1.0; and 3.0 mg/ml. These doses were calculated on a body weight basis extrapolated from the therapeutic dosage recommended by the company. Cells incubated in the absence of alliin served as controls. Culture media were collected at 24 h (for IL-1b, IL-6 and TNFa), or at 48 h for IL-2, the cells were removed by centrifugation and the supernatants were kept at ÿ708C until assayed. Cytokine concentration in the supernatants was tested using ELISA kits speci®cally for
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human IL-1b, IL-6, TNFa (Genzyme Corporation, Cambridge, MA) and IL-2 (R&D systems, Minneapolis, MN, USA) as detailed in the guide-line provided by the manufacturer. The detection levels of these cytokines in the assays were 30 pg/ml for IL-1b, IL-2 and TNFa and 15 pg/ml for IL-6. 2.4. Mitogen response Aliquots of PBMC (0.1 ml) suspension (2 106 ml) were divided into each well of 96 well plates (¯at bottom, Nunc) containing 0.1 ml of CM or 2% phytohemagglutinin (PHA-M, Difco), 10 mg/ml concanavalin A (Con A), or 20 mg/ml pokeweed mitogen (PWM, Sigma). Alliin was added at the onset of the culture at concentrations ranging between 0.5 and 3.0 mg/ ml. Cultures incubated in the absence of alliin served as controls. Triplicates were incubated for 3 d. Eighteen hours before harvesting, 3H-TdR (Thymidine-methyl-3H,4 Ci/mmole, ICN), 0.5 mCi/well, was added to the culture media. Radioactivity was measured with a LKB liquid scintillation counter model 3380. 2.5. Superoxide anion production Superoxide anion generation by PBMC and PMN cells was examined by the method of Johnston et al. [6]. Brie¯y, 4 106 cells suspended in 1 ml of PBS supplemented with 1 gr% glucose, 0.5 mM CaCl2 and 80 nM cytochrome C (from horse heart, Sigma, St. Louis, MD, USA) were incubated at 378C for 60 min without or with 2 10ÿ3 mM phorbol meristate acetate (PMA, Sigma) and without and with 3.0 mg/ml of alliin. After centrifugation at 1500 g, the supernatants were collected and the absorbency was determined at 550 nm. The amount of superoxide anions produced was directly proportional to the amount of reduced cytochrome C, calculated by subtracting the OD values without PMA, from those obtained with PMA. The speci®city of the reaction was con®rmed by complete inhibition of cytochrome C reduction following the addition of superoxide dismutase to the incubation mixture. 2.6. Phagocytosis Uniform polystyrene latex particles (0.1 ml of 5% suspension) (0.8 mm in diameter, Difco, Detroit, MI) in PBS were added to 106 ml of PMN cells incubated without and with 3.0 mg/ml of alliin. After 60 min the cells were sedimented, ®xed in cold 1% glutaraldehyde in phosphate buer pH 7.4, post®xed with osmium tetroxide and embedded in agar-100 resin (Agar Scienti®c LTD Cambridge, UK). Semi-thick sections cut with a LKB ultratome III were stained with methylene blue and the cells which engulfed latex particles, as well as the number of particles phagocytized by each individual cell were counted using a light microscope. At least 200 cells from each individual were examined. 2.7. Statistical evaluation Statistical analysis was carried out using repeated measures and paired t-test within each group. Results are expressed as mean2 SEM.
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3. Results
3.1. Mitogen induced proliferation (Fig 1) Concentrations of alliin between 0.5 and 3.0 mg/ml signi®cantly reduced the proliferative response of PBMC to Con A in a dose dependent manner down to 30% for the maximal concentration used. Alliin at concentrations of 1 and 3.0 mg/ml stimulated cell proliferation induced by PWM by 39 and 45%, respectively. Alliin had no eect on PHA-induced cell proliferation.
3.2. Cytokine production Incubation of PBMC with alliin at the three concentrations used in the study induced a signi®cant increase in spontaneous IL-1b production up to 30% when 3.0 mg/ml were applied. In LPS-treated cells an increase was observed only with the highest concentration used (14%, Fig. 2). Alliin did not have any eect on IL-2 production. As for its eect on IL-6, only the highest concentration used in the experiments caused a signi®cant decrease in both spontaneous and LPS-induced production by 22 and 30%, respectively (Fig. 3). There was no dierence in TNFa generation by LPS stimulated and non-stimulated cells incubated with 1 mg/ ml of alliin.
Fig. 1. Eect of various concentrations of alliin on mitogen induced cell proliferation. p < 0.04
p < 0.01.
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Fig. 2. Eect of dierent concentrations of alliin on spontaneous and LPS-induced IL-1b production. p < 0.05 p < 0.01 p < 0.001.
3.3. Oxygen superoxide production Alliin, at all three concentrations applied in the study did not exert any signi®cant eect on by both PMN and PBMC, the values being, 10.320.8 and 7.9 20.8 nm/106 cells, respectively, without alliin, vs 9.42 0.6 and 7.4 20.9 nm/106 following incubation with 3.0 mg/ml of alliin. Oÿ 2 -production
Fig. 3. Eect of alliin on spontaneous and LPS-induced IL-6 production. p < 0.05
p < 0.001.
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3.4. Phagocytosis Incubation of PMN cells with alliin caused a signi®cant increase in both the percentage of phagocyting cells, from 48.322.8 to 56.821.8% ( p < 0.02) and the number of latex particles engulfed by each individual cell, from 9.2 20.8 to 14.521.0 particles/cell ( p < 0.002, Fig. 4).
4. Discussion For many years, garlic has been recognized as a valuable folk medicine with a favorable eect against a large number of pathological conditions [2,23]. Powdered material obtained from garlic bulbs contains about 1% of alliin, which in the presence of the enzyme alliinase converts to allicin. It is accepted that the bene®cial properties of the garlic are due to allicin [22] and its transformation products which include ajoenes, vinyldithines, and polysul®des. Lachmann et al. [11] compared the pharmacokinetics of S35 labeled alliin, allicin and vinyldithines orally administered to rats and found that the blood activity pro®le for alliin diered considerably from that for allicin and vinyldithines. In their studies the maximum blood level for alliin was reached within 10 min and eliminated almost completely from the blood after 6 h. As for the remaining two garlic constituents, the maximum blood values were 30±60 min for allicin and 120 min for vinyldithines and values of more than 1000 ng-Eq/ml persisted at the end of the study after 72 h. While both allicin and alliin were shown to possess signi®cant antioxidant activity, the biological eect of allicin is attributed to its rapid reaction with thiol containing proteins [22], whereas that of alliin is due to its capacity to scavenge hydroxyl radicals [10,21].
Fig. 4. Percent of phagocyting cells and number of latex particles engulfed by each individual cell without and with alliin. p < 0.02 p < 0.002.
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The long list of conditions which may be prevented and treated with garlic and its compounds has brought about the widespread belief that garlic possesses a great therapeutic potential including immunostimulation [2,23]. The results of the present study showed that alliin in vitro exerts a certain eect on the immune response of peripheral blood cells. This eect consisted of an increase in PWM induced cell proliferation, spontaneous production of IL-1b, as well as an increase in both number of phagocyting cells and latex particles engulfed by each individual cell. Alliin caused a decrease in Con A induced cell proliferation and IL-6 production, but did not have any eect on PHA induced cell proliferation, IL-2 and superoxide anion generation. Since PWM is known to be a B-cell stimulator and an inducer of immunoglobulin synthesis, the increased proliferative response to PWM suggests an enhancing eect of alliin on B-lymphocytes for antibody production. The decreased response to Con-A, an activator of a subset of suppressor cells, indicates a possible immunomodulatory eect of alliin via reduction of suppressor cells. T cells activated with Con A may either suppress or stimulate B cells to proliferate or dierentiate to plasma cells [13]. Using anti-Tac, a speci®c monoclonal antibody, Con A induced stimulation of two types of human T cells, i.e. Tac (+) with and Tac (ÿ) without suppressive activity [27,28]. Moreover, mitogen stimulation of T cells was shown to be associated with membranal changes in various cell phenotypes resulting in an alteration of their susceptibility to drugs [9,16]. Therefore, the possibility that alliin exerts dierent eects on various cell populations comprising the PBMC cannot be excluded. Following activation with PHA, certain populations of resting T cells enter cell cycle and divide rapidly to produce large numbers of progenitors that will dierentiate into armed eector T cells. IL-2 produced by activated T cells drives T cell proliferation and dierentiation. The present ®ndings indicate that alliin aects speci®c lymphocyte subsets, but not T cells responding to PHA. The observed alteration in IL-1b and IL-6 production, which are known as proin¯ammatory cytokines, suggest an eect of alliin on the in¯ammatory reactions. Previous reports con®rm the role of garlic derivatives as immunomodulators. Morioka et al. [17] have reported that F4, a protein fraction isolated from aged garlic extract enhanced the cytotoxicity of human lymphocytes against natural killer (NK) sensitive K562 and NK-resistant M14 cell lines. This eect further increased when F4 was administered together with suboptimal doses of IL-2, a ®nding suggesting the existence of an enhancing eect of IL-2 on F4. An augmentation of Tlymphocyte blastogenesis by garlic compounds was reported by Lau et al. [12] indicating that garlic possesses a bene®cial immuno-stimulatory function. Romano et al. [24] have shown that ajoene, a compound isolated from garlic ethanolic extracts exerted a strong inhibitory eect on PHA, phorbol myristate and anti-CD3 induced proliferation of human lymphocytes, as well as LPS-induced TNF generation. Although our results did not show any eect of alliin on PHA induced proliferation, the stimulation of lymphocyte proliferation induced by PWM and the inhibition caused by Con A con®rm the eect of garlic extracts on lymphocyte membranedependent functions. The dierence between Romano's et al. [24] and our ®ndings might be due to the dierent forms of garlic preparations used in the studies. The stimulation of the phagocytic activity in the present study is of interest and further con®rms the value of garlic preparation for disease prevention. Increased phagocytic and immune functions induced by garlic preparation has been reported also by Sumiyoshi [26].
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