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Nitric oxide and tumor necrosis factor-α production by Ixeris dentata in mouse peritoneal macrophages
Journal of Ethnopharmacology 82 (2002) 217 /222 www.elsevier.com/locate/jethpharm
Nitric oxide and tumor necrosis factor-a production by Ixeris dentata in mouse peritoneal macrophages Hwan-Suck Chung a, Hyun-Ja Jeong a, Mi-Jung Han a, Seung-Taeck Park b, Kang-Kyung Seong c, Seung-Hwa Baek d, Dong-Myong Jeong e, Myong Jo Kim f, Hyung-Min Kim a,* a
Department of Oriental Pharmacy, College of Pharmacy, Korea Institute of Oriental Pharmacy, Wonkwang University, Iksan, Chonbuk 570-749, South Korea b Department of Anatomy, Wonkwang University School of Medicine, Iksan, Chonbuk 570-749, South Korea c College of Oriental Medicine, Wonkwang University, Iksan, Chonbuk 570-749, South Korea d Department of Herbal Resources, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Chonbuk 570-749, South Korea e Department of Electronic Engineering, Wonkwang University, Iksan, Chonbuk 570-749, South Korea f College of Agricultural and Life Science, Kangwon National University, Chuncheon 200-701, South Korea Accepted 4 July 2002
Abstract Using mouse peritoneal macrophages, we have examined the mechanism by which Ixeris dentata (IXD) regulates nitric oxide (NO) production. When IXD was used in combination with recombinant interferon-g (rIFN-g), there was a marked cooperative induction of NO production. However, IXD had no effect on NO production by itself. The increased production of NO from rIFNg plus IXD-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kB). Furthermore, treatment with IXD alone or rIFN-g plus IXD in peritoneal macrophages caused a significant increase in tumor necrosis factor-a (TNF-a) production. PDTC decreased TNF-a production induced by IXD significantly. These findings demonstrate that IXD increases the production of NO and TNF-a by rIFN-g-primed macrophages and suggest that NF-kB plays a critical role in mediating these effects of IXD. # 2002 Published by Elsevier Science Ireland Ltd. Keywords: Ixeris dentata ; Nitric oxide; Peritoneal macrophages; Tumor necrosis factor-a
1. Introduction The whole plant of Ixeris dentata (IXD, Asteraceae), a typical Oriental herb, has been used for treatment of indigestion, pneumonia, hepatitis, contusion and tumor (Kim and Lee, 1988; Kim, 1995). IXD is known to have aliphatics, triterpenoids and sesquiterpene glycosides in its composition (Arai et al., 1963; Seto et al., 1986). Nitric oxide (NO) is a highly reactive molecule produced from a guanidino nitrogen of arginine by NO synthase (NOS) enzymes (Nathan, 1992). Over the past decade, NO as a potent macrophage-derived
* Corresponding author. Tel.: /82-63-850-6805; fax: /82-63-8433421 E-mail address: [email protected] (H.-M. Kim).
effector molecule against a variety of bacteria, parasites, and tumors has received increasing attention (Nathan and Hibbs, 1991; Stuehr et al., 1989). Original evidence of tumor cell cytostasis and cytotoxity was found in macrophage-tumor cell co-cultures in which cytokine and/or lipopolysaccharides (LPS) stimulated macrophages inhibited metabolic functioning of co-cultured tumor cells (Hibbs et al., 1987). The expression of NO has been linked with DNA damage, thus stimulating the expression of wild-type p53 and ultimately leading to apoptosis (Messmer et al., 1994). NO may also be induced in target cells themselves yielding apoptotic cell death induced by autoexpression of iNOS (Hiess et al., 1994). Thus, NO mediated tumoricidal effects, similar to its antimicrobial effects, requires antigen recognition but results in antigen-nonspecific killing.
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The proinflammatory cytokine, tumor necrosis factor-a (TNF-a) regulates systemic responses to microbial infection or tissue injury (Akira et al., 1993; Tracey and Cerami, 1994). These signals stimulate immune functions and induce expression of acute phase reactants in the liver, among other effects. Production of TNF-a protein was enhanced by the presence of interferon-g (IFN-g). TNF-a then acted as an autocrine signal to amplify IFN-g-induced production of NO in macrophages (Lee et al., 1998). Macrophages are a major source of cytokine such as TNF-a, and induction of cytokine gene expression by LPS occurs primarily at the level of transcription and involves the action of several transcription factors, including members of the nuclear factor-kB (NF-kB)/ rel, C/EBP, Ets, and AP-1 protein families (Sweet and Hume, 1996). Especially, NF-kB bound to specific consensus DNA element present on the promoter of target genes initiates the transcription of TNF-a, iNOS, cyclo-oxygenase-2 and IL-6 (Kuprash et al., 1995). In the present study, we show that IXD synergistically induces the NO and TNF-a production by peritoneal macrophages when the cells are treated by recombinant IFN-g (rIFN-g). To investigate the mechanism of IXDinduced NO and TNF-a production, we examined the ability of NF-kB inhibitor such as pyrrolidine dithiocarbamate (PDTC) to block IXD-induced effect. PDTC decreased the NO and TNF-a production that had been induced by rIFN-g plus IXD. These findings may explain that IXD influences on NO and TNF-a production via the NF-kB signaling pathway.
chased from Dae Han Experimental Animal Centre (Eumsung, Republic of Korea). 2.2. Peritoneal macrophage cultures TG-elicited macrophages were harvested 3/4 days after i.p. injection of 2.5 ml TG to the mice and isolated, as reported previously (Narumi et al., 1990). Using 8 ml of HBSS containing 10 U/ml heparin, peritoneal lavage was performed. Then, the cells were distributed in DMEM, which was supplemented with 10% heatinactivated FBS, in 4-well tissue culture plates (2.5 / 105 cells per well) incubated for 3 h at 37 8C in an atmosphere of 5% CO2, washed three times with HBSS to remove non-adherent cells, and equilibrated with DMEM that contained 10% FBS before treatment. 2.3. Preparation of IXD The IXD were collected at Gaya Mountain (Kyungbuk, Korea) and authenticated by Professor J.M. Kim, Wonkwang University. A voucher specimen (number 01-02-12) was deposited at the Herbarium of the College of Pharmacy, Wonkwang University. The green sap extract of IXD (about 330 g) was prepared by mashing the 1 kg of raw herbs with a mortar. The extract was filtered, lyophilized, and kept at 4 8C. The yield of dried extract from green sap of IXD was about 12%. The powdered extract was dissolved in sterile saline and then filtered through 0.22 mm syringe filter. 2.4. Assay for endotoxin determination
2. Materials and methods 2.1. Reagents Murine rIFN-g (1 /106 U/ml) was purchased from Genzyme (Mu¨nchen, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), N -(1-naphtyl)-ethylenediamine dihydrochloride, LPS, sodium nitrite and PDTC were purchased from Sigma (St Louis, MO). Rabbit polyclonal antisera to iNOS was obtained from Transduction Laboratories (Lexington, KY). Recombinant TNF-a (rTNF-a), biotinylated TNF-a and anti-murine TNF-a were purchased from R&D system Inc, USA. NG-monomethyl-L-arginine (NGMMA) was purchased from Calbiochem (San Diego, CA). Thioglycollate (TG) was purchased from Difco Laboratories (Detroit, MI). 0.2 mm syringe filter and tissue culture plates of 96 wells, four wells and 100 mm diameter dishes were purchased from Nunc (Naperville, IL). DMEM containing Larginine (84 mg/l), Hank’s balanced salt solution (HBSS), fetal bovine serum (FBS) and other tissue culture reagents were purchased from Life Technologies (Grand Island, NY). Male C57BL/6 mice were pur-
IXD extracts used in this experiment was found to be free of endotoxin as determined within the limits of assay E-TOXATE kit (Sigma), performed according to manufacturer’s protocol. 2.5. Measurement of nitrite concentration Peritoneal macrophages (2.5 /105 cells per well) were cultured with rIFN-g (10 U/ml) for 6 h. The cells were then stimulated with various concentrations of IXD. NO synthesis in cell cultures was measured by a microplate assay method, as previously described (Xie et al., 1992). To measure nitrite, 100 ml aliquots were removed from conditioned medium and incubated with an equal volume of Griess reagent (1% sulfanilamide/ 0.1% N -(1-naphtyl)-ethylenediamine dihydrochloride/ 2.5% H3PO4) at room temperature for 10 min. The absorbance at 540 nm was determined in a Titertek Multiskan (Flow Laboratories, North Ryde, Australia). NO2 was determined by using sodium nitrite as a standard. The cell-free medium alone contained 5 /8 mM of NO2. This value was determined in each experiment
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and subtracted from the value obtained from the medium with peritoneal macrophages. 2.6. Western blot analysis Peritoneal macrophages (5 /106 cells per well) were incubated for 6 h with rIFN-g (10 U/ml). The cells were then stimulated with IXD (100 mg/ml) or LPS (10 mg/ml) for 12 h. Whole cell lysates were made by boiling peritoneal macrophages in sample buffer (62.5 mM Tris /HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 20% glycerol, and 10% 2-mercaptoethanol). Proteins in the cell lysates were then separated by 7% SDSpolyacrylamide gel electrophoresis and transferred to nitrocellulose paper. The membrane was then blocked with 5% skim milk in phosphate-buffered saline (PBS)tween-20 for 1 h at room temperature and then incubated with anti-iNOS antibodies. After washing in PBS-tween-20 three times, the blot was incubated with secondary antibody for 30 min and the antibody-specific proteins were visualized by the enhanced chemiluminesence detection system according to the recommended procedure (Amersham Corp. Newark, NJ). 2.7. Assay of TNF-a release Peritoneal macrophages (2.5 /105 cells per well) were incubated with rIFN-g (10 U/ml), IXD, rIFN-g plus LPS (10 mg/ml) and rIFN-g plus various concentrations of IXD for 24 h. Then the amount of TNF-a secreted by the cells was measured by a modified enzyme-linked immunosorbent assay (ELISA), as described previously (Kim et al., 1998). The ELISA was devised by coating 96-well plates of murine monoclonal antibody with specificity for TNF-a. Before use and between subsequent steps in the assay, coated plates were washed with PBS containing 0.05% tween-20. All reagents used in this assay were incubated for 2 h at 37 8C. The rTNF-a was diluted and used as a standard. Serial dilutions starting from 1 pg/ml were used to establish the standard curve. Assay plates were exposed sequentially to alkaline-phosphatase-conjugated goat anti-rabbit IgG. Optical density readings were made within 10 min of the substrate on a Titertek Multiskan with a 405 nm filter. Appropriate specificity controls were included.
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2.9. Statistical analysis Results were expressed as the mean9/S.E.M. of independent experiments, and statistical analysis was performed by the Student’s t-test to express the difference between two groups.
3. Results
3.1. Effects of IXD on NO production in activated peritoneal macrophages To determine the effect of IXD on the production of NO by mouse peritoneal macrophages, we treated nonprimed (resting) and rIFN-g-primed cells with IXD. The resultant NO production was determined by detecting nitrite concentrations in the cell supernatants after 48 h of treatment. As shown in Fig. 1, IXD had no effect on NO production in resting mouse peritoneal macrophages. However, when mouse peritoneal macrophages were primed for 6 h with murine rIFN-g and then treated with IXD, NO production was increased compared with that of non-primed conditions.
3.2. Effects of IXD on rIFN-g-primed iNOS expression Data in Fig. 2 show the effects of rIFN-g plus IXD treatments on the expression of iNOS protein in mouse peritoneal macrophages. rIFN-g plus IXD increased synergistically the expression of iNOS protein in mouse peritoneal macrophages.
2.8. High-performance liquid chromatography (HPLC) analysis The chromatographic system consisted of a pump (Waters 510 intelligent pump), a UV detector (Waters 484 UV detector), and Waters column oven. A mbondapack C-18 column (3.9 /300 mm, Waters) was used. An isocratic of 65% methanol was used as the mobile phase at a flow rate of 1.0 ml/min. The HPLC was monitored at 285 nm.
Fig. 1. Dose-dependent effects of IXD on NO synthesis in rIFN-gtreated peritoneal macrophages. Peritoneal macrophages (2.5/105 cells per well) were cultured with rIFN-g (10 U/ml). The peritoneal macrophages were then stimulated with various concentrations of IXD for 6 h after incubation. After 48 h of culture, NO release was measured by the Griess method (nitrite). Values are presented as the mean9/S.E.M. of three independent experiments duplicated in each run. *P B/0.05, **P B/ 0.001 compared with rIFN-g alone.
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Fig. 2. Effects of IXD on the expression of iNOS by rIFN-g plus IXDinduced peritoneal macrophages. The cells (5/106 cells per well) were incubated for 6 h with rIFN-g (10 U/ml). The cells were then stimulated with IXD (100 mg/ml) or LPS (10 mg/ml) for 12 h. The protein extracts were prepared, and then the samples were analyzed for iNOS expression by Western blotting as described in the method. 1, control; 2, rIFN-g; 3, rIFN-g/LPS; 4, rIFN-g/IXD.
3.3. Inhibition of IXD-induced NO production by NGMMA NGMMA is the specific inhibitor of NO production in the L-arginine-dependent pathway (Takao et al., 1997). To determine if the signaling mechanism in IXDinduced NO production participates in the L-argininedependent pathway in mouse peritoneal macrophages, the cells were incubated for 6 h in the presence of rIFN-g plus NGMMA. The production of nitrite by rIFN-g plus IXD in mouse peritoneal macrophages was progressively inhibited with increasing amount of NGMMA. The IXD-induced accumulation of nitrite was significantly blocked by NGMMA (0.1 /10 mM) (Fig. 3).
3.4. Effects of IXD on rIFN-g-induced TNF-a production We examined the synergistic cooperative effect of IXD on rIFN-g-induced TNF-a production. Mouse peritoneal macrophages secreted very low levels of TNF-a after 24 h of incubation with medium alone or rIFN-g alone. However, IXD increased TNF-a production in a dose-dependent manner and IXD plus rIFN-g increased TNF-a more (Fig. 4).
Fig. 4. Effects of IXD on the production of TNF-a in peritoneal macrophages. The cells (2.5/105 cells per well) were stimulated with or without rIFN-g (10 U/ml) plus various concentrations of IXD. The amount of TNF-a was measured by ELISA method after 24 h of incubation. Values are the mean9/S.E.M. of three independent experiments duplicated in each run. *P B/0.05 compared with medium alone, **PB/ 0.05 compared with rIFN-g alone.
3.5. Inhibition of IXD-induced NO and TNF-a production by PDTC It is known that PDTC, an anti-oxidant compound, inhibits NF-kB activation (Schreck et al., 1992). As an approach to define the signaling mechanism of IXD on NO production, we examined the influence of NF-kB inhibitor, PDTC, in rIFN-g plus IXD (100 mg/ml)treated mouse peritoneal macrophages. Adding PDTC (100 mM) to the rIFN-g plus IXD-treated mouse peritoneal macrophages decreased the synergistic effects of IXD on NO production significantly (Table 1). Many actions of TNF-a can be ascribed to its ability to activate NF-kB (Schwenger et al., 1998). Adding PDTC (100 mM) to the rIFN-g plus IXD (100 mg/ml)treated cells decreased the synergistic effects of IXD on TNF-a production significantly (Table 1). 3.6. Standardization of IXD Finally, we standardized IXD by HPLC. The structures of scutellarin and isoquercitrin were analyzed by a combination of EI-MS, 1H-NMR and 13C-NMR (data not shown). HPLC indicated that the resulting MeOH extracts of IXD contained were scutellarin (Rt 1.79 min) and isoquercitrin (Rt 3.66 min) (Fig. 5).
4. Discussion
G
Fig. 3. Effects of N MMA on IXD-induced nitrite accumulation in the cultured medium of peritoneal macrophages. The cells (2.5/105 cells per well) were incubated for 6 h with rIFN-g (10 U/ml) plus various concentrations of NGMMA. The cells were then treated with IXD (100 mg/ml) and cultured for 48 h. NO release was measured by the Griess method (nitirite). Values are presented as the mean9/S.E.M. of three independent experiments duplicated in each run. *P B/0.05, **P B/ 0.001 compared with control (absence of NGMMA).
In this study, we demonstrated that NO production in mouse peritoneal macrophages by IXD could be highly stimulated in combination with rIFN-g. IXD had a maximal effect on NO production at a concentration of 10 mg/ml in rIFN-g-treated mouse peritoneal macrophages. The results of this study suggest that IXD may provide a second signal for synergistic induction of NO production in mouse peritoneal macrophages.
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Table 1 Effects of PDTC on rIFN-g plus IXD-induced NO and TNF-a production in peritoneal macrophages Additiona rIFN-g (10 U/ml)
IXD (100 mg/ml)
PDTC (100 mM)
Nitrite concentration (mM)b
TNF-a secretion (ng/ml)c
4.5390.26 10.1090.60 47.3393.00 6.4090.55*
0.3490.01 1.0390.46 20.1691.82 1.9890.32*
Values are the mean9S.E.M. of three independent experiments duplicated in each run. *P B 0.01 compared with rIFN-g plus IXD. a Peritoneal macrophages (2.5 105 cells per well) were stimulated with various agents. b The amount of nitrite was measured after 48 h by the Griess method. c The amount of TNF-a was measured by ELISA method after 24 h of incubation.
Fig. 5. HPLC chromatogram of IXD. The detection was made at 285 nm.
NGMMA, an analog of L-arginine, inhibited rIFN-g plus IXD-induced NO production in peritoneal macrophages. The effective concentration of NGMMA for NO inhibition without any toxicity on cells is up to 10 mM, which is the concentration commonly used. The strong inhibition of nitrite production by NGMMA indicates that it is likely to depend upon NOS. At present, the precise physiological significance of NO and TNF-a production by IXD is unknown. However, over the past decade, TNF-a, an endogenous factor with tumor-selective cytotoxicity, has been recognized as an important host defense molecule that affects tumor cells. Moreover, the induction of NO and TNF-a production and gene expression by activated macrophages can lead to cytostatic and cytotoxic effects on malignant cells (Duerksen-Hughes et al., 1992; Balkwill et al., 1990; Gorelik et al., 1996). Because of the pivotal role of NO and TNF-a in the antimicrobial and tumoricidal activities of macrophages, significant effort has been focused on developing therapeutic agents that regulate NO and TNF-a production (Poderoso et al., 1999).
Signal transduction pathway of NO production has been previously reported as LPS stimulation of rIFN-gprimed macrophages induces NF-kB activation (Kim et al., 1995). NF-kB is now known to be ubiquitously expressed and to play a major role in controlling the expression of protein involved in immune, inflammatory and acute phase responses (Baeuerle and Henkel, 1994; Sha, 1998). Expression of iNOS and TNF-a genes is dependent on the activation of NF-kB (Albert and Baldwin, 1996). We found that the addition of NF-kB inhibitor, PDTC, inhibits the synergistic effect of IXD with rIFN-g on NO and TNF-a production. These results suggest that IXD increases NO and TNF-a production through NF-kB activation. The NF-kB system may provide a future target in cures for tumors. In conclusion, our results demonstrated that IXD acted as an accelerator of peritoneal macrophages activation by rIFN-g via a process involving L-arginine-dependent NO production and that it increased the production of TNF-a significantly via NF-kB activation. Although the precise mechanism of IXD to promote NO and TNF-a production induced by rIFNg remains to be further elucidated, NO and TNF-a production by IXD might explain its beneficial effect in the treatment of tumors.
Acknowledgements This work was supported by a grant of the Korea Health 21 R&D Project, the Ministry of Health & Welfare, Republic of Korea (HMP-00-PT-04-0006), and partially by Wonkwang University in 2002.
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Nitric oxide and tumor necrosis factor-a production by Ixeris dentata in mouse peritoneal macrophages Hwan-Suck Chung a, Hyun-Ja Jeong a, Mi-Jung Han a, Seung-Taeck Park b, Kang-Kyung Seong c, Seung-Hwa Baek d, Dong-Myong Jeong e, Myong Jo Kim f, Hyung-Min Kim a,* a
Department of Oriental Pharmacy, College of Pharmacy, Korea Institute of Oriental Pharmacy, Wonkwang University, Iksan, Chonbuk 570-749, South Korea b Department of Anatomy, Wonkwang University School of Medicine, Iksan, Chonbuk 570-749, South Korea c College of Oriental Medicine, Wonkwang University, Iksan, Chonbuk 570-749, South Korea d Department of Herbal Resources, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Chonbuk 570-749, South Korea e Department of Electronic Engineering, Wonkwang University, Iksan, Chonbuk 570-749, South Korea f College of Agricultural and Life Science, Kangwon National University, Chuncheon 200-701, South Korea Accepted 4 July 2002
Abstract Using mouse peritoneal macrophages, we have examined the mechanism by which Ixeris dentata (IXD) regulates nitric oxide (NO) production. When IXD was used in combination with recombinant interferon-g (rIFN-g), there was a marked cooperative induction of NO production. However, IXD had no effect on NO production by itself. The increased production of NO from rIFNg plus IXD-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kB). Furthermore, treatment with IXD alone or rIFN-g plus IXD in peritoneal macrophages caused a significant increase in tumor necrosis factor-a (TNF-a) production. PDTC decreased TNF-a production induced by IXD significantly. These findings demonstrate that IXD increases the production of NO and TNF-a by rIFN-g-primed macrophages and suggest that NF-kB plays a critical role in mediating these effects of IXD. # 2002 Published by Elsevier Science Ireland Ltd. Keywords: Ixeris dentata ; Nitric oxide; Peritoneal macrophages; Tumor necrosis factor-a
1. Introduction The whole plant of Ixeris dentata (IXD, Asteraceae), a typical Oriental herb, has been used for treatment of indigestion, pneumonia, hepatitis, contusion and tumor (Kim and Lee, 1988; Kim, 1995). IXD is known to have aliphatics, triterpenoids and sesquiterpene glycosides in its composition (Arai et al., 1963; Seto et al., 1986). Nitric oxide (NO) is a highly reactive molecule produced from a guanidino nitrogen of arginine by NO synthase (NOS) enzymes (Nathan, 1992). Over the past decade, NO as a potent macrophage-derived
* Corresponding author. Tel.: /82-63-850-6805; fax: /82-63-8433421 E-mail address: [email protected] (H.-M. Kim).
effector molecule against a variety of bacteria, parasites, and tumors has received increasing attention (Nathan and Hibbs, 1991; Stuehr et al., 1989). Original evidence of tumor cell cytostasis and cytotoxity was found in macrophage-tumor cell co-cultures in which cytokine and/or lipopolysaccharides (LPS) stimulated macrophages inhibited metabolic functioning of co-cultured tumor cells (Hibbs et al., 1987). The expression of NO has been linked with DNA damage, thus stimulating the expression of wild-type p53 and ultimately leading to apoptosis (Messmer et al., 1994). NO may also be induced in target cells themselves yielding apoptotic cell death induced by autoexpression of iNOS (Hiess et al., 1994). Thus, NO mediated tumoricidal effects, similar to its antimicrobial effects, requires antigen recognition but results in antigen-nonspecific killing.
0378-8741/02/$ - see front matter # 2002 Published by Elsevier Science Ireland Ltd. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 1 8 8 - 5
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The proinflammatory cytokine, tumor necrosis factor-a (TNF-a) regulates systemic responses to microbial infection or tissue injury (Akira et al., 1993; Tracey and Cerami, 1994). These signals stimulate immune functions and induce expression of acute phase reactants in the liver, among other effects. Production of TNF-a protein was enhanced by the presence of interferon-g (IFN-g). TNF-a then acted as an autocrine signal to amplify IFN-g-induced production of NO in macrophages (Lee et al., 1998). Macrophages are a major source of cytokine such as TNF-a, and induction of cytokine gene expression by LPS occurs primarily at the level of transcription and involves the action of several transcription factors, including members of the nuclear factor-kB (NF-kB)/ rel, C/EBP, Ets, and AP-1 protein families (Sweet and Hume, 1996). Especially, NF-kB bound to specific consensus DNA element present on the promoter of target genes initiates the transcription of TNF-a, iNOS, cyclo-oxygenase-2 and IL-6 (Kuprash et al., 1995). In the present study, we show that IXD synergistically induces the NO and TNF-a production by peritoneal macrophages when the cells are treated by recombinant IFN-g (rIFN-g). To investigate the mechanism of IXDinduced NO and TNF-a production, we examined the ability of NF-kB inhibitor such as pyrrolidine dithiocarbamate (PDTC) to block IXD-induced effect. PDTC decreased the NO and TNF-a production that had been induced by rIFN-g plus IXD. These findings may explain that IXD influences on NO and TNF-a production via the NF-kB signaling pathway.
chased from Dae Han Experimental Animal Centre (Eumsung, Republic of Korea). 2.2. Peritoneal macrophage cultures TG-elicited macrophages were harvested 3/4 days after i.p. injection of 2.5 ml TG to the mice and isolated, as reported previously (Narumi et al., 1990). Using 8 ml of HBSS containing 10 U/ml heparin, peritoneal lavage was performed. Then, the cells were distributed in DMEM, which was supplemented with 10% heatinactivated FBS, in 4-well tissue culture plates (2.5 / 105 cells per well) incubated for 3 h at 37 8C in an atmosphere of 5% CO2, washed three times with HBSS to remove non-adherent cells, and equilibrated with DMEM that contained 10% FBS before treatment. 2.3. Preparation of IXD The IXD were collected at Gaya Mountain (Kyungbuk, Korea) and authenticated by Professor J.M. Kim, Wonkwang University. A voucher specimen (number 01-02-12) was deposited at the Herbarium of the College of Pharmacy, Wonkwang University. The green sap extract of IXD (about 330 g) was prepared by mashing the 1 kg of raw herbs with a mortar. The extract was filtered, lyophilized, and kept at 4 8C. The yield of dried extract from green sap of IXD was about 12%. The powdered extract was dissolved in sterile saline and then filtered through 0.22 mm syringe filter. 2.4. Assay for endotoxin determination
2. Materials and methods 2.1. Reagents Murine rIFN-g (1 /106 U/ml) was purchased from Genzyme (Mu¨nchen, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), N -(1-naphtyl)-ethylenediamine dihydrochloride, LPS, sodium nitrite and PDTC were purchased from Sigma (St Louis, MO). Rabbit polyclonal antisera to iNOS was obtained from Transduction Laboratories (Lexington, KY). Recombinant TNF-a (rTNF-a), biotinylated TNF-a and anti-murine TNF-a were purchased from R&D system Inc, USA. NG-monomethyl-L-arginine (NGMMA) was purchased from Calbiochem (San Diego, CA). Thioglycollate (TG) was purchased from Difco Laboratories (Detroit, MI). 0.2 mm syringe filter and tissue culture plates of 96 wells, four wells and 100 mm diameter dishes were purchased from Nunc (Naperville, IL). DMEM containing Larginine (84 mg/l), Hank’s balanced salt solution (HBSS), fetal bovine serum (FBS) and other tissue culture reagents were purchased from Life Technologies (Grand Island, NY). Male C57BL/6 mice were pur-
IXD extracts used in this experiment was found to be free of endotoxin as determined within the limits of assay E-TOXATE kit (Sigma), performed according to manufacturer’s protocol. 2.5. Measurement of nitrite concentration Peritoneal macrophages (2.5 /105 cells per well) were cultured with rIFN-g (10 U/ml) for 6 h. The cells were then stimulated with various concentrations of IXD. NO synthesis in cell cultures was measured by a microplate assay method, as previously described (Xie et al., 1992). To measure nitrite, 100 ml aliquots were removed from conditioned medium and incubated with an equal volume of Griess reagent (1% sulfanilamide/ 0.1% N -(1-naphtyl)-ethylenediamine dihydrochloride/ 2.5% H3PO4) at room temperature for 10 min. The absorbance at 540 nm was determined in a Titertek Multiskan (Flow Laboratories, North Ryde, Australia). NO2 was determined by using sodium nitrite as a standard. The cell-free medium alone contained 5 /8 mM of NO2. This value was determined in each experiment
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and subtracted from the value obtained from the medium with peritoneal macrophages. 2.6. Western blot analysis Peritoneal macrophages (5 /106 cells per well) were incubated for 6 h with rIFN-g (10 U/ml). The cells were then stimulated with IXD (100 mg/ml) or LPS (10 mg/ml) for 12 h. Whole cell lysates were made by boiling peritoneal macrophages in sample buffer (62.5 mM Tris /HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 20% glycerol, and 10% 2-mercaptoethanol). Proteins in the cell lysates were then separated by 7% SDSpolyacrylamide gel electrophoresis and transferred to nitrocellulose paper. The membrane was then blocked with 5% skim milk in phosphate-buffered saline (PBS)tween-20 for 1 h at room temperature and then incubated with anti-iNOS antibodies. After washing in PBS-tween-20 three times, the blot was incubated with secondary antibody for 30 min and the antibody-specific proteins were visualized by the enhanced chemiluminesence detection system according to the recommended procedure (Amersham Corp. Newark, NJ). 2.7. Assay of TNF-a release Peritoneal macrophages (2.5 /105 cells per well) were incubated with rIFN-g (10 U/ml), IXD, rIFN-g plus LPS (10 mg/ml) and rIFN-g plus various concentrations of IXD for 24 h. Then the amount of TNF-a secreted by the cells was measured by a modified enzyme-linked immunosorbent assay (ELISA), as described previously (Kim et al., 1998). The ELISA was devised by coating 96-well plates of murine monoclonal antibody with specificity for TNF-a. Before use and between subsequent steps in the assay, coated plates were washed with PBS containing 0.05% tween-20. All reagents used in this assay were incubated for 2 h at 37 8C. The rTNF-a was diluted and used as a standard. Serial dilutions starting from 1 pg/ml were used to establish the standard curve. Assay plates were exposed sequentially to alkaline-phosphatase-conjugated goat anti-rabbit IgG. Optical density readings were made within 10 min of the substrate on a Titertek Multiskan with a 405 nm filter. Appropriate specificity controls were included.
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2.9. Statistical analysis Results were expressed as the mean9/S.E.M. of independent experiments, and statistical analysis was performed by the Student’s t-test to express the difference between two groups.
3. Results
3.1. Effects of IXD on NO production in activated peritoneal macrophages To determine the effect of IXD on the production of NO by mouse peritoneal macrophages, we treated nonprimed (resting) and rIFN-g-primed cells with IXD. The resultant NO production was determined by detecting nitrite concentrations in the cell supernatants after 48 h of treatment. As shown in Fig. 1, IXD had no effect on NO production in resting mouse peritoneal macrophages. However, when mouse peritoneal macrophages were primed for 6 h with murine rIFN-g and then treated with IXD, NO production was increased compared with that of non-primed conditions.
3.2. Effects of IXD on rIFN-g-primed iNOS expression Data in Fig. 2 show the effects of rIFN-g plus IXD treatments on the expression of iNOS protein in mouse peritoneal macrophages. rIFN-g plus IXD increased synergistically the expression of iNOS protein in mouse peritoneal macrophages.
2.8. High-performance liquid chromatography (HPLC) analysis The chromatographic system consisted of a pump (Waters 510 intelligent pump), a UV detector (Waters 484 UV detector), and Waters column oven. A mbondapack C-18 column (3.9 /300 mm, Waters) was used. An isocratic of 65% methanol was used as the mobile phase at a flow rate of 1.0 ml/min. The HPLC was monitored at 285 nm.
Fig. 1. Dose-dependent effects of IXD on NO synthesis in rIFN-gtreated peritoneal macrophages. Peritoneal macrophages (2.5/105 cells per well) were cultured with rIFN-g (10 U/ml). The peritoneal macrophages were then stimulated with various concentrations of IXD for 6 h after incubation. After 48 h of culture, NO release was measured by the Griess method (nitrite). Values are presented as the mean9/S.E.M. of three independent experiments duplicated in each run. *P B/0.05, **P B/ 0.001 compared with rIFN-g alone.
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Fig. 2. Effects of IXD on the expression of iNOS by rIFN-g plus IXDinduced peritoneal macrophages. The cells (5/106 cells per well) were incubated for 6 h with rIFN-g (10 U/ml). The cells were then stimulated with IXD (100 mg/ml) or LPS (10 mg/ml) for 12 h. The protein extracts were prepared, and then the samples were analyzed for iNOS expression by Western blotting as described in the method. 1, control; 2, rIFN-g; 3, rIFN-g/LPS; 4, rIFN-g/IXD.
3.3. Inhibition of IXD-induced NO production by NGMMA NGMMA is the specific inhibitor of NO production in the L-arginine-dependent pathway (Takao et al., 1997). To determine if the signaling mechanism in IXDinduced NO production participates in the L-argininedependent pathway in mouse peritoneal macrophages, the cells were incubated for 6 h in the presence of rIFN-g plus NGMMA. The production of nitrite by rIFN-g plus IXD in mouse peritoneal macrophages was progressively inhibited with increasing amount of NGMMA. The IXD-induced accumulation of nitrite was significantly blocked by NGMMA (0.1 /10 mM) (Fig. 3).
3.4. Effects of IXD on rIFN-g-induced TNF-a production We examined the synergistic cooperative effect of IXD on rIFN-g-induced TNF-a production. Mouse peritoneal macrophages secreted very low levels of TNF-a after 24 h of incubation with medium alone or rIFN-g alone. However, IXD increased TNF-a production in a dose-dependent manner and IXD plus rIFN-g increased TNF-a more (Fig. 4).
Fig. 4. Effects of IXD on the production of TNF-a in peritoneal macrophages. The cells (2.5/105 cells per well) were stimulated with or without rIFN-g (10 U/ml) plus various concentrations of IXD. The amount of TNF-a was measured by ELISA method after 24 h of incubation. Values are the mean9/S.E.M. of three independent experiments duplicated in each run. *P B/0.05 compared with medium alone, **PB/ 0.05 compared with rIFN-g alone.
3.5. Inhibition of IXD-induced NO and TNF-a production by PDTC It is known that PDTC, an anti-oxidant compound, inhibits NF-kB activation (Schreck et al., 1992). As an approach to define the signaling mechanism of IXD on NO production, we examined the influence of NF-kB inhibitor, PDTC, in rIFN-g plus IXD (100 mg/ml)treated mouse peritoneal macrophages. Adding PDTC (100 mM) to the rIFN-g plus IXD-treated mouse peritoneal macrophages decreased the synergistic effects of IXD on NO production significantly (Table 1). Many actions of TNF-a can be ascribed to its ability to activate NF-kB (Schwenger et al., 1998). Adding PDTC (100 mM) to the rIFN-g plus IXD (100 mg/ml)treated cells decreased the synergistic effects of IXD on TNF-a production significantly (Table 1). 3.6. Standardization of IXD Finally, we standardized IXD by HPLC. The structures of scutellarin and isoquercitrin were analyzed by a combination of EI-MS, 1H-NMR and 13C-NMR (data not shown). HPLC indicated that the resulting MeOH extracts of IXD contained were scutellarin (Rt 1.79 min) and isoquercitrin (Rt 3.66 min) (Fig. 5).
4. Discussion
G
Fig. 3. Effects of N MMA on IXD-induced nitrite accumulation in the cultured medium of peritoneal macrophages. The cells (2.5/105 cells per well) were incubated for 6 h with rIFN-g (10 U/ml) plus various concentrations of NGMMA. The cells were then treated with IXD (100 mg/ml) and cultured for 48 h. NO release was measured by the Griess method (nitirite). Values are presented as the mean9/S.E.M. of three independent experiments duplicated in each run. *P B/0.05, **P B/ 0.001 compared with control (absence of NGMMA).
In this study, we demonstrated that NO production in mouse peritoneal macrophages by IXD could be highly stimulated in combination with rIFN-g. IXD had a maximal effect on NO production at a concentration of 10 mg/ml in rIFN-g-treated mouse peritoneal macrophages. The results of this study suggest that IXD may provide a second signal for synergistic induction of NO production in mouse peritoneal macrophages.
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Table 1 Effects of PDTC on rIFN-g plus IXD-induced NO and TNF-a production in peritoneal macrophages Additiona rIFN-g (10 U/ml)
IXD (100 mg/ml)
PDTC (100 mM)
Nitrite concentration (mM)b
TNF-a secretion (ng/ml)c
4.5390.26 10.1090.60 47.3393.00 6.4090.55*
0.3490.01 1.0390.46 20.1691.82 1.9890.32*
Values are the mean9S.E.M. of three independent experiments duplicated in each run. *P B 0.01 compared with rIFN-g plus IXD. a Peritoneal macrophages (2.5 105 cells per well) were stimulated with various agents. b The amount of nitrite was measured after 48 h by the Griess method. c The amount of TNF-a was measured by ELISA method after 24 h of incubation.
Fig. 5. HPLC chromatogram of IXD. The detection was made at 285 nm.
NGMMA, an analog of L-arginine, inhibited rIFN-g plus IXD-induced NO production in peritoneal macrophages. The effective concentration of NGMMA for NO inhibition without any toxicity on cells is up to 10 mM, which is the concentration commonly used. The strong inhibition of nitrite production by NGMMA indicates that it is likely to depend upon NOS. At present, the precise physiological significance of NO and TNF-a production by IXD is unknown. However, over the past decade, TNF-a, an endogenous factor with tumor-selective cytotoxicity, has been recognized as an important host defense molecule that affects tumor cells. Moreover, the induction of NO and TNF-a production and gene expression by activated macrophages can lead to cytostatic and cytotoxic effects on malignant cells (Duerksen-Hughes et al., 1992; Balkwill et al., 1990; Gorelik et al., 1996). Because of the pivotal role of NO and TNF-a in the antimicrobial and tumoricidal activities of macrophages, significant effort has been focused on developing therapeutic agents that regulate NO and TNF-a production (Poderoso et al., 1999).
Signal transduction pathway of NO production has been previously reported as LPS stimulation of rIFN-gprimed macrophages induces NF-kB activation (Kim et al., 1995). NF-kB is now known to be ubiquitously expressed and to play a major role in controlling the expression of protein involved in immune, inflammatory and acute phase responses (Baeuerle and Henkel, 1994; Sha, 1998). Expression of iNOS and TNF-a genes is dependent on the activation of NF-kB (Albert and Baldwin, 1996). We found that the addition of NF-kB inhibitor, PDTC, inhibits the synergistic effect of IXD with rIFN-g on NO and TNF-a production. These results suggest that IXD increases NO and TNF-a production through NF-kB activation. The NF-kB system may provide a future target in cures for tumors. In conclusion, our results demonstrated that IXD acted as an accelerator of peritoneal macrophages activation by rIFN-g via a process involving L-arginine-dependent NO production and that it increased the production of TNF-a significantly via NF-kB activation. Although the precise mechanism of IXD to promote NO and TNF-a production induced by rIFNg remains to be further elucidated, NO and TNF-a production by IXD might explain its beneficial effect in the treatment of tumors.
Acknowledgements This work was supported by a grant of the Korea Health 21 R&D Project, the Ministry of Health & Welfare, Republic of Korea (HMP-00-PT-04-0006), and partially by Wonkwang University in 2002.
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