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Immunomodulatory activity of polysaccharides isolated from Salicornia herbacea
International Immunopharmacology 6 (2006) 1451 – 1458 www.elsevier.com/locate/intimp
Immunomodulatory activity of polysaccharides isolated from Salicornia herbacea Sun-A Im a , Kyungjae Kim b , Chong-Kil Lee a,⁎ a
College of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea b Department of Pharmacy, SahmYook University, Seoul 139-742, South Korea
Received 13 December 2005; received in revised form 3 January 2006; accepted 10 April 2006
Abstract Several types of immunomodulatory polysaccharides originated from plants or mushrooms have been used as immunotherapeutic agents in the treatment of cancers. Here, we describe an immunomodulatory polysaccharide that cannot only activate monocytic cells strongly, but also induce differentiation of monocytic cells into macrophages. High molecular weight substances, SHE, were isolated from Salicornia herbacea, which has been used to treat a variety of diseases including cancers in traditional oriental remedy. The immunomodulatory activities of SHE were examined on a mouse monocytic cell line, RAW 264.7 cells. We found that SHE activated RAW cells to produce cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and nitric oxide (NO) dose dependently. SHE also induced the expression of co-stimulatory molecules such as B7-1 and CD40, and increased phagocytic activity on opsonized sheep red blood cells. While increasing these parameters of macrophage activation, SHE inhibited the growth of RAW cells dose dependently inducing morphological changes from slightly adherent monocytic cells to strongly adherent macrophages. The active components of SHE appeared to be polysaccharides, and not an endotoxin. These results show that polysaccharides originated from S. herbacea possess potent immunomodulatory activity on monocyte/ macrophage lineage cells. © 2006 Elsevier B.V. All rights reserved. Keywords: Salicornia herbacea; Polysaccharides; Immunomodulator; Macrophage
1. Introduction Salicornia herbacea has no leaves, but is formed of cylindrical branches of a light green color. It grows naturally in the western coast of Korean peninsula, especially in the salt mashes and on the muddy seashores. Its growth in Japan is so rare that it has been designated as a natural monument in Japan in ⁎ Corresponding author. Tel.: +82 43 261 2826; fax: +82 43 268 2732. E-mail address: [email protected] (C.-K. Lee). 1567-5769/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2006.04.011
1921. In fork medicine, S. herbacea has been used to treat a variety of diseases such as constipation, obesity, diabetes and cancers. In recent years, patents were even filed claiming that S. herbacea was effective in the amelioration of inflammatory responses and in the prevention of atherosclerosis, hypertension and tumors [1–3]. The biological mechanisms for these activities, however, have not been elucidated, nor the active components. Being a halophyte, S. herbacea contains large amounts of salt and minerals, especially calcium, magnesium and iodine. It also contains large amounts
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of betaine and choline [4]. The beneficial effects of consuming S. herbacea may in part due to betaine or choline absorption. Betaine has been shown to reduce potentially toxic level of homocysteine, an amino acid found normally in the body, and a high level of homocysteine has been implicated to increase the chance of developing heart disease, stroke, liver disease, and peripheral vascular disease [5,6]. Choline is a major component of phospholipid and acetylcholine [7,8]. S. herbacea has also been reported to contain small molecular weight substances such as isorhammetin-3O-β-glucoside, which inhibits rat lens aldolase reductase [9], and tungtungmadic acid, which has antioxidant activity [10]. In the present study, we examined the immunomodulatory activity of S. herbacea extract, SHE, on a monocytic cell line, RAW 264.7 cells. We found that SHE activated several parameters of macrophage activation such as morphological changes, nitric oxide and cytokine production, expression of co-stimulatory molecules, and phagocytic activity. The active component of SHE appeared to be polysaccharides. The fact that S. herbacea contains immunomodulatory polysaccharides may explain some of the therapeutic efficacies of S. herbacea, which has been used in folk medicine to treat various diseases including cancers.
removal of precipitates by centrifugation. Sugar composition was analyzed as described previously [13]. Briefly, SHE was hydrolyzed with 2 M trifluoroacetic acid for 4 h at 100°C. The hydrolysates were analyzed by high pH anion exchange HPLC on a Bio-LC DX-300 carbohydrate analyzer system (Dionex, Sunnyvale, CA, USA) using a CarboPac PA1 anion-exchange column (4.5 × 250mm, Dionex, Sunnyvale, CA, USA) attached with PED2 detector (Dionex). The mobile phase was isocratic solvent of 16 mM NaOH. 2.3. Cell culture RAW cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 100U/ml penicillin and 100 μg/ ml streptomycin (Invitrogen), and 50 μM 2-mercptoethanol (Invitrogen) at 37 °C, 5% CO2 condition. 2.4. Proliferation assay RAW cells were cultured in the presence of different concentrations of SHE in a 96-well microtiter plate (2 × 104 cells/well). DNA synthesis was measured by 3Hthymidine (DuPont) incorporation (0.5 μCi/well) for the final 6 h of the 2-day culture period. The cells were harvested onto glass fiber filter paper using a cell harvester. The filters were washed, dried, and then counted in a microbeta liquid scintillation counter (Wallac, USA). 2.5. Cytokine production
2. Material and methods
S. herbacea was collected in September 2001 from Boryeong, Chungnam Province, South Korea. A voucher specimen was deposited at the College of Pharmacy, Chungbuk National University.
RAW cells were cultured in the presence of different concentrations of SHE in a 24-well microtiter plate (5 × 105 cells/well) in a total volume of 1 ml. After 48 h incubation, the culture supernatants were collected, and the amounts of IL-1β and TNF-α were measured using commercial immunoassay kits (R&D System) according to the manufacturer's instruction.
2.2. Preparation and characterization of SHE
2.6. Nitric oxide production
The dried whole plant of S. herbacea was homogenized, and then extracted with boiling water twice for 3 h. The combined supernatant was concentrated to a small volume in vacuo at 40 °C and precipitated with 3 volumes of ethanol at 4 °C overnight. The precipitate was collected by centrifugation, washed with 70% ethanol, lyophilized, and dissolved in phosphate buffered saline (PBS). Total carbohydrate and protein contents were determined by phenol-sulfuric acid method [11] and by bicinchoninic acid assay kit (Pierce, Rockford, IL), respectively. Proteins contained in SHE were destroyed by treatment with protease [12]. Briefly, SHE (50 mg) was dissolved in 50 ml of 50 mM Tris–HCl, pH 7.9, containing 10 mM CaCl2, and pronase (Sigma) was added (50 mg). The reaction mixture was incubated at 37 °C overnight, and then dialyzed against distilled water for 3 days. The non-dialyzable portion was lyophilized after
RAW cells were cultured in the presence of different concentrations of SHE in a 24-well microtiter plate (5 × 105 cells/well) in a total volume of 1 ml. After 48 h stimulation, 50 μl of cell-free supernatant were collected, incubated with 50 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, 0.5% H3PO4) at room temperature for 5 min, and the absorbance at 550nm was determined in a Dynatech MR500 microplate reader. The concentration of NO−2 was determined from a least squares linear regression analysis of a sodium nitrite standard curve.
2.1. Plant material
2.7. Semi-quantitative RT-PCR RAW cells were cultured in the presence of 100 μg/ml SHE in 6-well tissue culture plate (1 × 106 cells/well) for 6h. Total RNA (1 μg) isolated from the stimulated cells was
S.-A. Im et al. / International Immunopharmacology 6 (2006) 1451–1458
reverse transcribed into cDNA using an RT-PCR kit (AccuPowder RT/PCR Premix, Bioner Co., Daejeon, Korea). Primers used in the present study are as follows: inducible nitric oxide synthetase (iNOS) (forward 5′GTCAACTGCAAGAGAACGGAGAAC-3′ and reverse 5′GAGCTCCTCCAGAGGGTAGGCT-3′), TNF-α (forward 5′-CTTCAGCCCCAGCAGTGTATTCTTT-3′ and reverse 5′-AGAGAACCTGGGAGTAGACAAGGTA-3′), IL-1β (forward 5′-CTTCAGCCCCAGCAGTGTATTCTTT-3′ and reverse 5′-AGAGAACCTGGGAGTAGACAAGGTA-3′), and β-actin (forward 5′-CACCACACCTTCTACAATGAGCTGC-3′ and reverse 5′-GCTCAGGAGGAGCAATGATCTTGAT-3′). The cDNAs from the reverse transcription reactions were amplified under the following conditions: denaturation at 94 °C for 30 s, annealing at 56 °C (iNOS), 50 °C (TNF-α) or 60 °C (IL-1β) for 30 s, and extension at 72 °C for 90 s with a final extension at 72 °C for 5 min. Amplified cDNA products were resolved on 1.5% agarose gel by electrophoresis and then stained with ethidium bromide.
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3. Results 3.1. Effects of SHE on the growth of RAW cells In the first step to examine the immunomodulatory activity of SHE, we added SHE to the cultures of RAW cells, and the morphological changes were observed 2 days later. Microscopic observation of the cell morphology showed that almost all the cells cultured with SHE were enlarged with extensive cytoplasmic spreading, while total cell numbers were greatly decreased compared to that of control cultures (Fig. 1A). Growth inhibitory activity of SHE on RAW cells was further documented by 3H-thymidine uptake for the final 6 h of the 2day culture period. As shown in Fig. 1B, SHE inhibited the growth of RAW cells dose dependently. The growth inhibitory activity of SHE reached up to 90% at a concentration of 100μg/ml. The growth inhibitory activity of SHE was not due to direct cytotoxicity on the cells as shown in the following section of the present study.
2.8. Flow cytometry Cells were stained with monoclonal antibodies recognizing murine cell surface markers as described previously [14], and flow cytometric analysis was performed on a FACS Caliver (Becton-Dickinson). The monoclonal antibodies, anti-CD40 (clone 3/23), anti-ICAM-1 (clone 3E2), anti-IAb (clone AF6-120.1), anti-B7-1 (clone 16-10A1), anti-B7-2 (clone GL1), and isotype-matched control antibodies were purchased from Pharmingen (San Diego, CA). Dead cells were gated out by their low forward angle light scatter intensity. In most analysis, 10,000 cells were scored. 2.9. Phagocytic activity RAW cells were cultured in the presence of different concentrations of SHE for 48 h in slide chambers (1 × 105/ chamber) in a volume of 1.0ml, and then 100 μl/well of IgGopsonized sheep red blood cells (SRBCs, 5%) were added. The slide chambers were incubated for 1 h at 37 °C, rinsed with PBS and then treated with ACK lysis buffer for 3 min to lyse uningested SRBCs. Opsonized SRBCs were prepared by incubating SRBCs with a 1:256-diluted mouse anti-SRBC IgG antibody (Cordis Lab., Miami) for 30 min at 37 °C in a shaking water bath. 2.10. Removal of endotoxin Possible contaminants of endotoxin contained in SHE were removed using Affi-Prep Polymyxin Matrix (BIO-RAD). Briefly, 1 ml of Affi-Prep Polymyxin Matrix was packed in a Bio-spin column (BIO-RAD), centrifuged for 2 min at 200×g, and then 0.5 ml of SHE (200 μg/ml), LPS (4 μg/ml) or SHE (200 μg/ml) and LPS (4 μg/ml) mixture was added. After incubating overnight at 4 °C, the effluent was recovered from the column by centrifugation at the same condition.
Fig. 1. Effects of SHE on the growth of RAW cells. (A) RAW cells were cultured in the presence of 100μg/ml SHE for 2days, and the morphological changes were photographed at 100× magnification. (B) RAW cells were cultured in the presence of different concentrations of SHE (200, 50, 25, 12.5, 6.25, 3.13 and 1.6μg/ml) for 2 days, and DNA synthesis was measured by 3H-thymidine incorporation for the final 6h of the culture period of 2 days. The CPM values of untreated RAW cells served as control values in the calculation of % inhibition.
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3.2. Effects of SHE on the production of cytokines and nitric oxide To examine whether SHE-activated RAW cells produced cytokines, the culture supernatants were collected at 48 h, and the amounts of TNF-α and IL-1β were measured by ELISAs. As shown in Fig. 2, TNF-α and IL-1β were produced in a dose-dependent manner in response to SHE stimulation. Interestingly, SHE-activated RAW cells produced especially large amounts of TNF-α reaching approximately 7.5ng/ml at a SHE concentration of 100 μg/ml. The effective dose of SHE inducing 50% of maximum response (ED50) for TNF-α production was approximately 15 μg/ml. The ED50 for IL-1β production was approximately 40 μg/ ml. RAW cells also produced nitric oxide dose dependently with ED50 value of 4 μg/ml (Fig. 2C). To examine whether the stimulatory effects of SHE on cytokine and NO production were attributable to its influence on the expression of respective genes, semi-quantitative RTPCRs were performed for the mRNAs of iNOS, TNF-α and IL-1β. As shown in Fig. 3, the transcripts for iNOS, TNF-α and IL-1β were hardly detectable in unstimulated RAW cells. The amounts of iNOS, TNF-α and IL-1β transcripts (the lower bands) were, however, increased significantly upon exposure
Fig. 3. Effects of SHE on the expression of mRNAs of inducible nitric oxide synthetase (iNOS), TNF-α and IL-1β. RAW cells were cultured in the presence of SHE (100μg/ml) for 6h. Total RNA was isolated from the stimulated cells, and subjected to semi-quantitative RT-PCR. Transcripts of β-actin (the upper bands) were served as internal controls. A representative gel graph from 3 experiments was shown.
to SHE for 6 h. Transcripts of β-actin (the upper bands) were served as internal controls. 3.3. Effects of SHE on the expression of surface molecules Since increased expression of co-stimulatory molecules could be a marker of macrophage activation, we also examined
Fig. 2. Effects of SHE on the production of cytokines and nitric oxide. RAW cells were cultured in the presence of different concentrations of SHE (100, 50, 25, 12.5, 6.25, 3.13 and 1.6 μg/ml) for 2days, and the culture supernatants were assayed for TNF-α (A), IL-1β (B), and nitric oxide (C).
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Fig. 4. Phenotypic analysis of RAW cells stimulated with SHE. RAW cells were cultured in the presence of 50 μg/ml SHE for 2days, and the cells were collected, washed, and then used for immunophenotypic analysis. Levels of expression (thin line) were illustrated in comparison to isotype control (dotted line).
the effects of SHE on the expression of co-stimulatory molecules on RAW cells. As shown in Fig. 4, SHE at a concentration of 50 μg/ml increased the expression of B7-1 and CD40 significantly. Expression of MHC class II molecule, which was not expressed on unstimulated RAW cells, was not increased by SHE stimulation. In contrast, expression of ICAM-1, which was constitutively expressed in unstimulated RAW cells in a high level, was increased further by SHE stimulation. 3.4. Effects of SHE on phagocytic activity One of the most distinguished features of macrophage activation would be an increase in phagocytic activity. Phagocytic activity of SHE-activated RAW cells was examined with opsonized SRBCs. As shown in Fig. 5, the average
number of SRBCs phagocytized by unstimulated RAW cells was 0.4, while the average number of SRBCs phagocytized by SHE-activated RAW cells was 5.2. 3.5. Composition and characterization of SHE SHE is a white powder readily soluble in water. It contained approximately 46% polysaccharide when determined by a phenol-sulfuric acid method using glucose as a reference sugar and less than 3.4% protein when determined by a bicinchoninic acid assay kit using bovine serum albumin as a reference protein. Complete hydrolysis of SHE with 2 M trifluoroacetic acid followed by monosaccharide composition analysis revealed that major sugars composing SHE are galactose, glucose and mannose. The average molar ratio of galactose, glucose and mannose was 5:1.5:1. To confirm that
Fig. 5. Phagocytic activity of RAW cells stimulated with SHE. RAW cells were cultured in the presence of 50μg/ml SHE for 2days, and IgGopsonized SRBCs were added. After 1h incubation at 37 °C, the plates were rinsed with PBS, and then uningested SRBCs were lysed with ACK lysis buffer. The macrophages were photographed at 400 × without staining.
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4. Discussion
Fig. 6. Effects of pronase treatment. Proteins contained in SHE were digested with pronase, and the remaining polysaccharides were lyophilized after extensive dialysis. The lyophilized polysaccharides were dissolved in PBS, and added to the cultures of RAW cells for 2days. The amounts of nitric oxide were measured using a Griess reagent.
the active component of SHE is a polysaccharide, proteins contained in SHE were destroyed by treatment with pronase. Enzymatic digestion of SHE with pronase followed by extensive dialysis decreased the protein content to 1.2%. The polysaccharide to protein ratio of pronase-treated SHE was 35:1, which was significantly different from that of untreated SHE (13.5:1). The immunomodulatory activity of the pronasetreated SHE was also examined in RAW cells. As shown in Fig. 6, pronase-treated SHE induced almost the same amounts of nitric oxide compared to untreated SHE. In order to examine whether SHE uses mannose receptor to activate RAW cells, we performed blocking experiments with mannose. Addition of mannose up to 5 mg/ml did not block macrophage-activating activity of SHE (data not shown).
The present study shows that S. herbacea contains immunomodulatory polysaccharides. SHE prepared from S. herbacea was shown to inhibit proliferation of RAW cells inducing morphological changes from slightly adherent monocytic cells into strongly adherent macrophages. In normal cultures, RAW cells tended to be round, and cells with cytoplasmic spreading appeared in a frequency of less than 5%. Because macrophages are end-stage cells that do not proliferate further, the growth inhibitory activity of SHE may be due to induction of further differentiation of monocytic RAW cells. Meanwhile, SHE was shown to stimulate RAW cells to produce TNF-α and IL-1β, which were often implicated as key mediators produced from macrophages in response to bacterial LPS, infection and inflammatory stimuli [15,16]. SHE was also shown to stimulate RAW cells to produce nitric oxide, which has been shown to be the principal effector molecule produced by macrophages for cytotoxic activity and can be used as a quantitative index of macrophage activation [17]. Immature macrophages do not induce primary immune responses because they do not express the required class II MHC molecules and co-stimulatory molecules. SHE was shown to enhance the expression of some of costimulatory molecules such as B7-1 and CD40, although it was unable to induce the expression of class II MHC molecules. SHE was also shown to activate phagocytic activity. These results demonstrate that SHE is an activator of macrophages.
3.6. Endotoxin contamination To ensure that the effects of SHE were not due to endotoxin contamination, SHE was treated with polymyxin B, and the immunomodulating activity was examined in RAW cells. As shown in Fig. 7, passage of LPS solution (4 μg/ml) with polymyxin B-affinity column reduced the nitric oxide-inducing activity almost completely, showing that polymyxin B-affinity column absorbed LPS almost completely. Passage of SHE solution (200 μg/ml) with polymyxin B-affinity column, however, did not reduce the nitric oxide-inducing activity in a significant level. To exclude possible interference of the complex-forming activity of polymyxin B by unknown substance(s) contained in the SHE solution, SHE was mixed with LPS, passed through polymyxin B-affinity column, and then the nitric oxideinducing activity in RAW cells was examined. As shown in Fig. 7, polymyxin B-affinity column could remove LPS from the mixture of SHE and LPS. These results indicate that the immunomodulatory activity of SHE was not due to LPS contamination.
Fig. 7. Effects of polymyxin B-treatment. LPS (4μg/ml), SHE (200μg/ ml), or LPS (4μg/ml) and SHE (200μg/ml) mixture was added to polymyxin B-affinity column, incubated overnight at 4°C, eluted from the column by centrifugation, and then added to the cultures of RAW cells (final, 1:80 dilution) for 2days. Filled bars represent nitric oxideinducing activity of the reagents before polymyxin B-treatment, and open bars represent nitric oxide-inducing activity of the same reagents after polymyxin B-treatment.
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The active component of SHE appeared to be polysaccharides, because treatment of SHE with pronase did not destroy macrophage-activating activity of SHE. The fact that S. herbacea contains immunomodulatory polysaccharides may explain some of the therapeutic efficacies of S. herbacea, which has long been used in folk medicine to treat various diseases including cancers. In fact, immunomodulatory polysaccharides have been implicated for the therapeutic efficacy of many plants or mushrooms such as Aloe vera [14,18], Angelica acutiloba [12], Acanthopanax koreanum [19], and Lentinus edodes [20]. The immunomodulatory polysaccharides isolated from these plants or mushrooms appeared to activate immune responses primarily by activation of macrophages, although direct activation of B cells and other immune cells has been implicated. For instance, acemannan, a major polysaccharide contained in the gel of A. vera, was shown to activate macrophages to produce inflammatory cytokines such as IL-6 and TNF-α [21], increase NO production by macrophages [22–24] and upregulate phagocytic and candidicidal activities of macrophages [25]. The chemical properties of the immunomodulatory polysaccharides contained in S. herbacea have not been studied yet. Composition analysis of the polysaccharide, however, suggests that it is quite different from wellknown immunomodulatory polysaccharides such as acemannan, lentinan and PSK [18–20]. Because endotoxin is a strong activator of macrophages and is contaminated in many plant materials, possible contamination of endotoxin is always a matter of concern for the high molecular weight components isolated from plants. The macrophage-activating activity of SHE, however, was not due to endotoxin contamination as shown by polymyxin B-treatment experiments. Macrophages are unique cells in the immune system in that they not only can initiate immune responses, but also can serve as effector cells. Furthermore, activated macrophages become more efficient antigen presenting cells because they express increased levels of class II MHC molecules and co-stimulatory molecules [26,27]. Thus, based on the results of the present study which showed that S. herbacea contains polysaccharides activating macrophages, we are tempted to speculate that some of the therapeutic efficacies such as anti-tumor activity of S. herbacea are due to the immunomodulatory polysaccharides. Acknowledgement This work was supported by the research grant of the Chungbuk National University in 2004.
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[24] Djeraba A, Quere P. In vivo macrophage activation in chickens with Acemannan, a complex carbohydrate extracted from Aloe vera. Int J Immunopharmacol 2000;22:365–72. [25] Stuart RW, Lefkowitz DL, Lincoln JA, Howard K, Gelderman MP, Lefkowitz SS, et al. Upregulation of phagocytosis and candidicidal activity of macrophages exposed to the immunostimulant acemannan. Int J Immunopharmacol 1997;19:75–82. [26] Adams DO, Hamilton TA. The cell biology of macrophage activation. Annu Rev Immunol 1984;2:283–318. [27] Fujiwara N, Kobayashi K. Macrophages in inflammation. Curr Drug Targets Inflamm Allergy 2005;4:281–6.
Immunomodulatory activity of polysaccharides isolated from Salicornia herbacea Sun-A Im a , Kyungjae Kim b , Chong-Kil Lee a,⁎ a
College of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea b Department of Pharmacy, SahmYook University, Seoul 139-742, South Korea
Received 13 December 2005; received in revised form 3 January 2006; accepted 10 April 2006
Abstract Several types of immunomodulatory polysaccharides originated from plants or mushrooms have been used as immunotherapeutic agents in the treatment of cancers. Here, we describe an immunomodulatory polysaccharide that cannot only activate monocytic cells strongly, but also induce differentiation of monocytic cells into macrophages. High molecular weight substances, SHE, were isolated from Salicornia herbacea, which has been used to treat a variety of diseases including cancers in traditional oriental remedy. The immunomodulatory activities of SHE were examined on a mouse monocytic cell line, RAW 264.7 cells. We found that SHE activated RAW cells to produce cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and nitric oxide (NO) dose dependently. SHE also induced the expression of co-stimulatory molecules such as B7-1 and CD40, and increased phagocytic activity on opsonized sheep red blood cells. While increasing these parameters of macrophage activation, SHE inhibited the growth of RAW cells dose dependently inducing morphological changes from slightly adherent monocytic cells to strongly adherent macrophages. The active components of SHE appeared to be polysaccharides, and not an endotoxin. These results show that polysaccharides originated from S. herbacea possess potent immunomodulatory activity on monocyte/ macrophage lineage cells. © 2006 Elsevier B.V. All rights reserved. Keywords: Salicornia herbacea; Polysaccharides; Immunomodulator; Macrophage
1. Introduction Salicornia herbacea has no leaves, but is formed of cylindrical branches of a light green color. It grows naturally in the western coast of Korean peninsula, especially in the salt mashes and on the muddy seashores. Its growth in Japan is so rare that it has been designated as a natural monument in Japan in ⁎ Corresponding author. Tel.: +82 43 261 2826; fax: +82 43 268 2732. E-mail address: [email protected] (C.-K. Lee). 1567-5769/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2006.04.011
1921. In fork medicine, S. herbacea has been used to treat a variety of diseases such as constipation, obesity, diabetes and cancers. In recent years, patents were even filed claiming that S. herbacea was effective in the amelioration of inflammatory responses and in the prevention of atherosclerosis, hypertension and tumors [1–3]. The biological mechanisms for these activities, however, have not been elucidated, nor the active components. Being a halophyte, S. herbacea contains large amounts of salt and minerals, especially calcium, magnesium and iodine. It also contains large amounts
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of betaine and choline [4]. The beneficial effects of consuming S. herbacea may in part due to betaine or choline absorption. Betaine has been shown to reduce potentially toxic level of homocysteine, an amino acid found normally in the body, and a high level of homocysteine has been implicated to increase the chance of developing heart disease, stroke, liver disease, and peripheral vascular disease [5,6]. Choline is a major component of phospholipid and acetylcholine [7,8]. S. herbacea has also been reported to contain small molecular weight substances such as isorhammetin-3O-β-glucoside, which inhibits rat lens aldolase reductase [9], and tungtungmadic acid, which has antioxidant activity [10]. In the present study, we examined the immunomodulatory activity of S. herbacea extract, SHE, on a monocytic cell line, RAW 264.7 cells. We found that SHE activated several parameters of macrophage activation such as morphological changes, nitric oxide and cytokine production, expression of co-stimulatory molecules, and phagocytic activity. The active component of SHE appeared to be polysaccharides. The fact that S. herbacea contains immunomodulatory polysaccharides may explain some of the therapeutic efficacies of S. herbacea, which has been used in folk medicine to treat various diseases including cancers.
removal of precipitates by centrifugation. Sugar composition was analyzed as described previously [13]. Briefly, SHE was hydrolyzed with 2 M trifluoroacetic acid for 4 h at 100°C. The hydrolysates were analyzed by high pH anion exchange HPLC on a Bio-LC DX-300 carbohydrate analyzer system (Dionex, Sunnyvale, CA, USA) using a CarboPac PA1 anion-exchange column (4.5 × 250mm, Dionex, Sunnyvale, CA, USA) attached with PED2 detector (Dionex). The mobile phase was isocratic solvent of 16 mM NaOH. 2.3. Cell culture RAW cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 100U/ml penicillin and 100 μg/ ml streptomycin (Invitrogen), and 50 μM 2-mercptoethanol (Invitrogen) at 37 °C, 5% CO2 condition. 2.4. Proliferation assay RAW cells were cultured in the presence of different concentrations of SHE in a 96-well microtiter plate (2 × 104 cells/well). DNA synthesis was measured by 3Hthymidine (DuPont) incorporation (0.5 μCi/well) for the final 6 h of the 2-day culture period. The cells were harvested onto glass fiber filter paper using a cell harvester. The filters were washed, dried, and then counted in a microbeta liquid scintillation counter (Wallac, USA). 2.5. Cytokine production
2. Material and methods
S. herbacea was collected in September 2001 from Boryeong, Chungnam Province, South Korea. A voucher specimen was deposited at the College of Pharmacy, Chungbuk National University.
RAW cells were cultured in the presence of different concentrations of SHE in a 24-well microtiter plate (5 × 105 cells/well) in a total volume of 1 ml. After 48 h incubation, the culture supernatants were collected, and the amounts of IL-1β and TNF-α were measured using commercial immunoassay kits (R&D System) according to the manufacturer's instruction.
2.2. Preparation and characterization of SHE
2.6. Nitric oxide production
The dried whole plant of S. herbacea was homogenized, and then extracted with boiling water twice for 3 h. The combined supernatant was concentrated to a small volume in vacuo at 40 °C and precipitated with 3 volumes of ethanol at 4 °C overnight. The precipitate was collected by centrifugation, washed with 70% ethanol, lyophilized, and dissolved in phosphate buffered saline (PBS). Total carbohydrate and protein contents were determined by phenol-sulfuric acid method [11] and by bicinchoninic acid assay kit (Pierce, Rockford, IL), respectively. Proteins contained in SHE were destroyed by treatment with protease [12]. Briefly, SHE (50 mg) was dissolved in 50 ml of 50 mM Tris–HCl, pH 7.9, containing 10 mM CaCl2, and pronase (Sigma) was added (50 mg). The reaction mixture was incubated at 37 °C overnight, and then dialyzed against distilled water for 3 days. The non-dialyzable portion was lyophilized after
RAW cells were cultured in the presence of different concentrations of SHE in a 24-well microtiter plate (5 × 105 cells/well) in a total volume of 1 ml. After 48 h stimulation, 50 μl of cell-free supernatant were collected, incubated with 50 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, 0.5% H3PO4) at room temperature for 5 min, and the absorbance at 550nm was determined in a Dynatech MR500 microplate reader. The concentration of NO−2 was determined from a least squares linear regression analysis of a sodium nitrite standard curve.
2.1. Plant material
2.7. Semi-quantitative RT-PCR RAW cells were cultured in the presence of 100 μg/ml SHE in 6-well tissue culture plate (1 × 106 cells/well) for 6h. Total RNA (1 μg) isolated from the stimulated cells was
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reverse transcribed into cDNA using an RT-PCR kit (AccuPowder RT/PCR Premix, Bioner Co., Daejeon, Korea). Primers used in the present study are as follows: inducible nitric oxide synthetase (iNOS) (forward 5′GTCAACTGCAAGAGAACGGAGAAC-3′ and reverse 5′GAGCTCCTCCAGAGGGTAGGCT-3′), TNF-α (forward 5′-CTTCAGCCCCAGCAGTGTATTCTTT-3′ and reverse 5′-AGAGAACCTGGGAGTAGACAAGGTA-3′), IL-1β (forward 5′-CTTCAGCCCCAGCAGTGTATTCTTT-3′ and reverse 5′-AGAGAACCTGGGAGTAGACAAGGTA-3′), and β-actin (forward 5′-CACCACACCTTCTACAATGAGCTGC-3′ and reverse 5′-GCTCAGGAGGAGCAATGATCTTGAT-3′). The cDNAs from the reverse transcription reactions were amplified under the following conditions: denaturation at 94 °C for 30 s, annealing at 56 °C (iNOS), 50 °C (TNF-α) or 60 °C (IL-1β) for 30 s, and extension at 72 °C for 90 s with a final extension at 72 °C for 5 min. Amplified cDNA products were resolved on 1.5% agarose gel by electrophoresis and then stained with ethidium bromide.
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3. Results 3.1. Effects of SHE on the growth of RAW cells In the first step to examine the immunomodulatory activity of SHE, we added SHE to the cultures of RAW cells, and the morphological changes were observed 2 days later. Microscopic observation of the cell morphology showed that almost all the cells cultured with SHE were enlarged with extensive cytoplasmic spreading, while total cell numbers were greatly decreased compared to that of control cultures (Fig. 1A). Growth inhibitory activity of SHE on RAW cells was further documented by 3H-thymidine uptake for the final 6 h of the 2day culture period. As shown in Fig. 1B, SHE inhibited the growth of RAW cells dose dependently. The growth inhibitory activity of SHE reached up to 90% at a concentration of 100μg/ml. The growth inhibitory activity of SHE was not due to direct cytotoxicity on the cells as shown in the following section of the present study.
2.8. Flow cytometry Cells were stained with monoclonal antibodies recognizing murine cell surface markers as described previously [14], and flow cytometric analysis was performed on a FACS Caliver (Becton-Dickinson). The monoclonal antibodies, anti-CD40 (clone 3/23), anti-ICAM-1 (clone 3E2), anti-IAb (clone AF6-120.1), anti-B7-1 (clone 16-10A1), anti-B7-2 (clone GL1), and isotype-matched control antibodies were purchased from Pharmingen (San Diego, CA). Dead cells were gated out by their low forward angle light scatter intensity. In most analysis, 10,000 cells were scored. 2.9. Phagocytic activity RAW cells were cultured in the presence of different concentrations of SHE for 48 h in slide chambers (1 × 105/ chamber) in a volume of 1.0ml, and then 100 μl/well of IgGopsonized sheep red blood cells (SRBCs, 5%) were added. The slide chambers were incubated for 1 h at 37 °C, rinsed with PBS and then treated with ACK lysis buffer for 3 min to lyse uningested SRBCs. Opsonized SRBCs were prepared by incubating SRBCs with a 1:256-diluted mouse anti-SRBC IgG antibody (Cordis Lab., Miami) for 30 min at 37 °C in a shaking water bath. 2.10. Removal of endotoxin Possible contaminants of endotoxin contained in SHE were removed using Affi-Prep Polymyxin Matrix (BIO-RAD). Briefly, 1 ml of Affi-Prep Polymyxin Matrix was packed in a Bio-spin column (BIO-RAD), centrifuged for 2 min at 200×g, and then 0.5 ml of SHE (200 μg/ml), LPS (4 μg/ml) or SHE (200 μg/ml) and LPS (4 μg/ml) mixture was added. After incubating overnight at 4 °C, the effluent was recovered from the column by centrifugation at the same condition.
Fig. 1. Effects of SHE on the growth of RAW cells. (A) RAW cells were cultured in the presence of 100μg/ml SHE for 2days, and the morphological changes were photographed at 100× magnification. (B) RAW cells were cultured in the presence of different concentrations of SHE (200, 50, 25, 12.5, 6.25, 3.13 and 1.6μg/ml) for 2 days, and DNA synthesis was measured by 3H-thymidine incorporation for the final 6h of the culture period of 2 days. The CPM values of untreated RAW cells served as control values in the calculation of % inhibition.
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3.2. Effects of SHE on the production of cytokines and nitric oxide To examine whether SHE-activated RAW cells produced cytokines, the culture supernatants were collected at 48 h, and the amounts of TNF-α and IL-1β were measured by ELISAs. As shown in Fig. 2, TNF-α and IL-1β were produced in a dose-dependent manner in response to SHE stimulation. Interestingly, SHE-activated RAW cells produced especially large amounts of TNF-α reaching approximately 7.5ng/ml at a SHE concentration of 100 μg/ml. The effective dose of SHE inducing 50% of maximum response (ED50) for TNF-α production was approximately 15 μg/ml. The ED50 for IL-1β production was approximately 40 μg/ ml. RAW cells also produced nitric oxide dose dependently with ED50 value of 4 μg/ml (Fig. 2C). To examine whether the stimulatory effects of SHE on cytokine and NO production were attributable to its influence on the expression of respective genes, semi-quantitative RTPCRs were performed for the mRNAs of iNOS, TNF-α and IL-1β. As shown in Fig. 3, the transcripts for iNOS, TNF-α and IL-1β were hardly detectable in unstimulated RAW cells. The amounts of iNOS, TNF-α and IL-1β transcripts (the lower bands) were, however, increased significantly upon exposure
Fig. 3. Effects of SHE on the expression of mRNAs of inducible nitric oxide synthetase (iNOS), TNF-α and IL-1β. RAW cells were cultured in the presence of SHE (100μg/ml) for 6h. Total RNA was isolated from the stimulated cells, and subjected to semi-quantitative RT-PCR. Transcripts of β-actin (the upper bands) were served as internal controls. A representative gel graph from 3 experiments was shown.
to SHE for 6 h. Transcripts of β-actin (the upper bands) were served as internal controls. 3.3. Effects of SHE on the expression of surface molecules Since increased expression of co-stimulatory molecules could be a marker of macrophage activation, we also examined
Fig. 2. Effects of SHE on the production of cytokines and nitric oxide. RAW cells were cultured in the presence of different concentrations of SHE (100, 50, 25, 12.5, 6.25, 3.13 and 1.6 μg/ml) for 2days, and the culture supernatants were assayed for TNF-α (A), IL-1β (B), and nitric oxide (C).
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Fig. 4. Phenotypic analysis of RAW cells stimulated with SHE. RAW cells were cultured in the presence of 50 μg/ml SHE for 2days, and the cells were collected, washed, and then used for immunophenotypic analysis. Levels of expression (thin line) were illustrated in comparison to isotype control (dotted line).
the effects of SHE on the expression of co-stimulatory molecules on RAW cells. As shown in Fig. 4, SHE at a concentration of 50 μg/ml increased the expression of B7-1 and CD40 significantly. Expression of MHC class II molecule, which was not expressed on unstimulated RAW cells, was not increased by SHE stimulation. In contrast, expression of ICAM-1, which was constitutively expressed in unstimulated RAW cells in a high level, was increased further by SHE stimulation. 3.4. Effects of SHE on phagocytic activity One of the most distinguished features of macrophage activation would be an increase in phagocytic activity. Phagocytic activity of SHE-activated RAW cells was examined with opsonized SRBCs. As shown in Fig. 5, the average
number of SRBCs phagocytized by unstimulated RAW cells was 0.4, while the average number of SRBCs phagocytized by SHE-activated RAW cells was 5.2. 3.5. Composition and characterization of SHE SHE is a white powder readily soluble in water. It contained approximately 46% polysaccharide when determined by a phenol-sulfuric acid method using glucose as a reference sugar and less than 3.4% protein when determined by a bicinchoninic acid assay kit using bovine serum albumin as a reference protein. Complete hydrolysis of SHE with 2 M trifluoroacetic acid followed by monosaccharide composition analysis revealed that major sugars composing SHE are galactose, glucose and mannose. The average molar ratio of galactose, glucose and mannose was 5:1.5:1. To confirm that
Fig. 5. Phagocytic activity of RAW cells stimulated with SHE. RAW cells were cultured in the presence of 50μg/ml SHE for 2days, and IgGopsonized SRBCs were added. After 1h incubation at 37 °C, the plates were rinsed with PBS, and then uningested SRBCs were lysed with ACK lysis buffer. The macrophages were photographed at 400 × without staining.
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4. Discussion
Fig. 6. Effects of pronase treatment. Proteins contained in SHE were digested with pronase, and the remaining polysaccharides were lyophilized after extensive dialysis. The lyophilized polysaccharides were dissolved in PBS, and added to the cultures of RAW cells for 2days. The amounts of nitric oxide were measured using a Griess reagent.
the active component of SHE is a polysaccharide, proteins contained in SHE were destroyed by treatment with pronase. Enzymatic digestion of SHE with pronase followed by extensive dialysis decreased the protein content to 1.2%. The polysaccharide to protein ratio of pronase-treated SHE was 35:1, which was significantly different from that of untreated SHE (13.5:1). The immunomodulatory activity of the pronasetreated SHE was also examined in RAW cells. As shown in Fig. 6, pronase-treated SHE induced almost the same amounts of nitric oxide compared to untreated SHE. In order to examine whether SHE uses mannose receptor to activate RAW cells, we performed blocking experiments with mannose. Addition of mannose up to 5 mg/ml did not block macrophage-activating activity of SHE (data not shown).
The present study shows that S. herbacea contains immunomodulatory polysaccharides. SHE prepared from S. herbacea was shown to inhibit proliferation of RAW cells inducing morphological changes from slightly adherent monocytic cells into strongly adherent macrophages. In normal cultures, RAW cells tended to be round, and cells with cytoplasmic spreading appeared in a frequency of less than 5%. Because macrophages are end-stage cells that do not proliferate further, the growth inhibitory activity of SHE may be due to induction of further differentiation of monocytic RAW cells. Meanwhile, SHE was shown to stimulate RAW cells to produce TNF-α and IL-1β, which were often implicated as key mediators produced from macrophages in response to bacterial LPS, infection and inflammatory stimuli [15,16]. SHE was also shown to stimulate RAW cells to produce nitric oxide, which has been shown to be the principal effector molecule produced by macrophages for cytotoxic activity and can be used as a quantitative index of macrophage activation [17]. Immature macrophages do not induce primary immune responses because they do not express the required class II MHC molecules and co-stimulatory molecules. SHE was shown to enhance the expression of some of costimulatory molecules such as B7-1 and CD40, although it was unable to induce the expression of class II MHC molecules. SHE was also shown to activate phagocytic activity. These results demonstrate that SHE is an activator of macrophages.
3.6. Endotoxin contamination To ensure that the effects of SHE were not due to endotoxin contamination, SHE was treated with polymyxin B, and the immunomodulating activity was examined in RAW cells. As shown in Fig. 7, passage of LPS solution (4 μg/ml) with polymyxin B-affinity column reduced the nitric oxide-inducing activity almost completely, showing that polymyxin B-affinity column absorbed LPS almost completely. Passage of SHE solution (200 μg/ml) with polymyxin B-affinity column, however, did not reduce the nitric oxide-inducing activity in a significant level. To exclude possible interference of the complex-forming activity of polymyxin B by unknown substance(s) contained in the SHE solution, SHE was mixed with LPS, passed through polymyxin B-affinity column, and then the nitric oxideinducing activity in RAW cells was examined. As shown in Fig. 7, polymyxin B-affinity column could remove LPS from the mixture of SHE and LPS. These results indicate that the immunomodulatory activity of SHE was not due to LPS contamination.
Fig. 7. Effects of polymyxin B-treatment. LPS (4μg/ml), SHE (200μg/ ml), or LPS (4μg/ml) and SHE (200μg/ml) mixture was added to polymyxin B-affinity column, incubated overnight at 4°C, eluted from the column by centrifugation, and then added to the cultures of RAW cells (final, 1:80 dilution) for 2days. Filled bars represent nitric oxideinducing activity of the reagents before polymyxin B-treatment, and open bars represent nitric oxide-inducing activity of the same reagents after polymyxin B-treatment.
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The active component of SHE appeared to be polysaccharides, because treatment of SHE with pronase did not destroy macrophage-activating activity of SHE. The fact that S. herbacea contains immunomodulatory polysaccharides may explain some of the therapeutic efficacies of S. herbacea, which has long been used in folk medicine to treat various diseases including cancers. In fact, immunomodulatory polysaccharides have been implicated for the therapeutic efficacy of many plants or mushrooms such as Aloe vera [14,18], Angelica acutiloba [12], Acanthopanax koreanum [19], and Lentinus edodes [20]. The immunomodulatory polysaccharides isolated from these plants or mushrooms appeared to activate immune responses primarily by activation of macrophages, although direct activation of B cells and other immune cells has been implicated. For instance, acemannan, a major polysaccharide contained in the gel of A. vera, was shown to activate macrophages to produce inflammatory cytokines such as IL-6 and TNF-α [21], increase NO production by macrophages [22–24] and upregulate phagocytic and candidicidal activities of macrophages [25]. The chemical properties of the immunomodulatory polysaccharides contained in S. herbacea have not been studied yet. Composition analysis of the polysaccharide, however, suggests that it is quite different from wellknown immunomodulatory polysaccharides such as acemannan, lentinan and PSK [18–20]. Because endotoxin is a strong activator of macrophages and is contaminated in many plant materials, possible contamination of endotoxin is always a matter of concern for the high molecular weight components isolated from plants. The macrophage-activating activity of SHE, however, was not due to endotoxin contamination as shown by polymyxin B-treatment experiments. Macrophages are unique cells in the immune system in that they not only can initiate immune responses, but also can serve as effector cells. Furthermore, activated macrophages become more efficient antigen presenting cells because they express increased levels of class II MHC molecules and co-stimulatory molecules [26,27]. Thus, based on the results of the present study which showed that S. herbacea contains polysaccharides activating macrophages, we are tempted to speculate that some of the therapeutic efficacies such as anti-tumor activity of S. herbacea are due to the immunomodulatory polysaccharides. Acknowledgement This work was supported by the research grant of the Chungbuk National University in 2004.
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