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The nitric oxide-producing properties of Solanum lyratum
Journal of Ethnopharmacology 67 (1999) 163 – 169 www.elsevier.com/locate/jethpharm
The nitric oxide-producing properties of Solanum lyratum H.M. Kim a,*, M.J. Kim a, E. Li b, Y.S. Lyu c, C.Y. Hwang c, N.H. An a b
a College of Pharmacy, Wonkwang Uni6ersity, Iksan, Chonbuk 570 -749, South Korea Department of Integration of Traditional and Western Medicine, Hebei Medical Uni6ersity, Hebei, People’s Republic of China c College of Oriental Medicine, Wonkwang Uni6ersity, Iksan, Chonbuk 570 -749, South Korea
Received 27 November 1998; received in revised form 11 January 1999; accepted 18 January 1999
Abstract We examined the effect of Solanum lyratum Thunb. (Solanaceae) (SL) on the production of nitric oxide (NO). Stimulation of mouse peritoneal macrophages with SL after the treatment of recombinant interferon-g (rIFN-g) resulted in increased NO synthesis. SL had no effect on NO synthesis by itself. When SL was used in combination with rIFN-g, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of SL on NO synthesis was shown 6 h after treatment with rIFN-g. The increased production of NO from rIFN-g plus SL-stimulated cells was decreased by the treatment with staurosporin. In addition, synergy between rIFN-g and SL was mainly dependent on SL-induced tumor necrosis factor-a (TNF-a) secretion. All the preparations of SL were endotoxin free. The present results indicate that the capacity of SL to increase NO production from rIFN-g-primed mouse peritoneal macrophages is the result of SL-induced TNF-a secretion via the signal transduction pathway of PKC activation. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Solanum lyratum; Nitric oxide; Mouse peritoneal macrophages; Recombinant interferon-g; Tumor necrosis factor-a
1. Introduction The Solanum lyratum Thunb (Solanaceae) (SL) herbs, well known as ‘Back-Mo-Deung’ in Korea, has been used for regulating body immune function and ability, and it still occupies an important place in traditional Korean medicine. This herb contains solalyrantines A and B, respectively, together with several furostanol, spirostanol and spirosolane glycosides (Lee et al., 1997). Nitric * Corresponding author. Tel.: +82-653-850-6805; fax: + 82-653-843-3421. E-mail address: [email protected] (H.M. Kim)
oxide (NO) is a highly reactive molecule produced from a guanidino nitrogen of L-arginine in a reaction catalyzed by a family of NO synthase (NOS) enzymes (Nathan, 1992). Recent studies have demonstrated the crucial role of NO in the antimicrobial and tumoricidal activities of murine macrophages (Moncada et al., 1991). More recent studies suggest that NO also has antiviral effects in both murine and human cells. Croen (1993) demonstrated that stimulation of a murine macrophage cell line with interferon-gamma (IFN-g) and lipopolysaccharide (LPS) resulted in high level NO production and a 1000-fold inhibition of herpes simplex virus-1 replication. Synthe-
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 1 1 - 2
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H.M. Kim et al. / Journal of Ethnopharmacology 67 (1999) 163–169
sis of high amounts of this reactive radical requires expression of inducible nitric oxide synthase (iNOS), and this initial step is tightly regulated (Nathan and Xie, 1994) in order to prevent potential damage to the host (Moncada and Higgs, 1993). For the production of NO in macrophages, IFN-g is required as a priming signal before those cells can subsequently be triggered by a second signal, for example, LPS, phorbol ester or tumor necrosis factor-a (TNF-a; Nathan, 1992; Lee et al., 1998). In this study, we show that SL synergistically induces NO synthesis by mouse peritoneal macrophages when the cells are treated by recombinant interferon-g (rIFN-g), but SL alone has no significant effect on the NO production. In addition, we will point out that SL has the potency to modulate NO production via the inhibition of protein kinase (PKC) activity, and that synergy between rIFN-g plus SL is mainly dependent on SL-induced TNF-a secretion.
2. Materials and methods
2.1. Reagents Murine rIFN-g (1 ×106 U/mg), recombinant tumor necrosis factor-a (rTNF-a: 1 ×105 U/ml) and rabbit anti-murine TNF-a antibody were purchased from Genzyme (Munchen, Germany). Dulbecco’s modified Eagle’s medium (DMEM), staurosporine (STSN), N-(1-naphthyl)-ethylenediamine dihydrochloride, sodium nitrite, and sulfanilamide were purchased from Sigma (St. Louis, MO). N G-monomethyl-L-arginine (N GMMA) was purchased from Calbiochem (Sandiego, CA). Thioglycollate (TG, fluid thioglycollate medium dehydrated) was purchased from Difco Laboratories (Detroit, MI). All reagents and media for tissue culture experiments were tested for their LPS content with use of a colorimetric Limulus amoebocyte lysate assay (detection limit, 10 pg/ ml; Whittaker Bioproducts, Walkersville, MD). None of these reagents contained endotoxins. Ninety-six-well tissue culture plates and 100-mm diameter petri dishes were purchased from Nunc (Naperville, IL). DMEM containing L-arginine
(84 mg/l), Hank’s balanced salt solution (HBSS), fetal calf serum (FCS), and other tissue culture reagents were purchased from Life Technologies (Grand Island, NY).
2.2. Animals Male C57BL/6 mice were purchased from Dae Han Experimental Animal Center (Eumsung, Korea). The animals were maintained at College of Pharmacy, Wonkwang University. The mice were housed five to ten per cage in a laminar air flow room maintained under a temperature of 229 1°C and relative humidity of 559 10% throughout the study.
2.3. Preparation of extracts The plant sample (Number, 4-96-35) was collected on a farm of Chonnam Province, South Korea. A voucher specimen was deposited at the Herbarium at the College of Pharmacy, Wonkwang University, and extracted with distilled water at 70°C for 5 h. The extract was filtered through a 0.45-mm filter and filtrate was lyophilized. The w/w yield of the extract was about 9.2%. The sample was identified by T.Y. Shin, College of Pharmacy, Woosuk University.
2.4. Cell cultures TG-elicited macrophages were harvested 3 days after i.p. injection of 2.5 ml TG (formula per liter; 15 g of Bacto Casitone, 5 g of Bacto yeast extract, 5.5 g of Bacto dextrose, 2.5 g of sodium chloride, 0.5 g of L-cystine, 0.5 g of sodium thioglycollate, 0.75 g of Bacto agar, 0.001 g of resazurin). We suspended 29.8 g in 1 l distilled water and boiled to dissolve completely and sterilized at 121− 124°C for 15 min and then supplied the prepared medium to mice 8–12 weeks of age, isolated as reported previously (Narumi et al., 1990). Peritoneal lavage was performed by using 8 ml of HBSS, which contained 10 U/ml heparin. Then, the cells were distributed in DMEM, which was supplemented with 10% (v/v) FCS, in either 96well tissue culture plates (2 ×105 cells/well) or 100-mm diameter plastic petri dishes (1×107
H.M. Kim et al. / Journal of Ethnopharmacology 67 (1999) 163–169
cells/dish), incubated for 3 h at 37°C in an atmosphere of 5% CO2, washed three times with HBSS to remove non-adherent cells, and equilibrated with DMEM that contained 10% FCS before treatment.
165
2%-azinobis. OD readings were made within 10 min of addition of the substrate on a Titertek Multiskan with a 405-nm filter. Appropriate specificity controls were included.
2.7. Statistical analysis 2.5. Measurement of nitrite and nitrate 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 supernatants of cultured cells and incubated with an equal volume of the Griess reagent (1% sulfanilamide/0.1% N-(1naphthyl)-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− (nitrite) was determined by using sodium nitrite as a standard. The cellfree medium alone contained 5 – 8 mM of nitrite; this value was determined in each experiment and subtracted from the value obtained from the medium with cells. In some experiments, NO3− (nitrate) was measured by reducing nitrate to nitrite with bacterial nitrate reductase measuring nitrite by using Griess reagent (Langerman et al., 1990).
2.6. Assay of TNF-a secretion TNF-a secretion was measured by modification of an ELISA, as previously described (Scuderi et al., 1986). The ELISA was sensitive to TNF-a concentrations in medium above 40 pg/ml. The ELISA was devised by coating 96-well plates with 6.25 ng/well of murine monoclonal antibody with specificity for human TNF-a. Before use and between subsequent steps in the assay, coated plates were washed twice with PBS containing 0.05% Tween 20 and twice with PBS alone. All reagents used in this assay were incubated for 1 h at room temperature with coated wells. For the standard curve, TNF-a was added to serum previously determined to be negative for endogenous TNF-a. After exposure to medium, assay plates were sequentially exposed to rabbit anti-TNF-a, phosphatase-conjugated goat anti-rabbit IgG, and 2,
Data collected were expressed as means9 S.E. Statistical analysis was performed by the Student’s t-test to express the difference between two groups.
3. Results
3.1. Effect of SL on rIFN-g-induced NO production Initially, we wished to determine whether mouse peritoneal macrophages could be stimulated by SL, either alone or in combination with rIFN-g, to induce NO production. Isolated mouse peritoneal macrophages were cultured either in medium alone or in medium that contained rIFNg (5 U/ml). Then, cells were stimulated with SL at various times during 48 h culture, and NO release was measured by using the Griess method. As Table 1 shows, apparently SL alone did not induce NO synthesis, whereas SL in combination with rIFN-g synergistically increased NO synthesis in cultured peritoneal macrophages. The maximum cooperative effect of SL for NO release were shown 6 h after rIFN-g treatment. The dose-dependent effects of SL in the presence of rIFN-g on NO synthesis were shown in Fig. 1. The synergistic effect was maximal at 1000 mg/ml of SL. Concentrations of less than 10 mg/ml were considerably less effective and, in some cases, ineffective.
3.2. Inhibition of SL-induced NO production by N GMMA, a competiti6e inhibitor of NO synthase To determine if the signaling mechanism in SL-induced NO production is involved in the L-arginine-dependent pathway in mouse peritoneal macrophages, the cells were incubated for 6 h in the presence of rIFN-g plus N GMMA. The production of nitrite by rIFN-g plus SL in mouse
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Table 1 Effect of SL on rIFN-g-induced NO production in mouse peritoneal macrophagesa Addition
Final concentration (mM)
rIFN-g
SL
LPS
NO− 2
− NO− 2 plus NO3
− + + − − + + +
− − − + +(0 h) +(0 h) +(6 h) +(12 h)
− − +(10 ng/ml) − − − − −
B5 13.5 92.8 45.4 99.4* 8.4 90.9 B5 44.3 93.3* 46.2 93.7* 39.5 95.1*
B5 28.3 94.1 86.5 921.6* 15.7 92.3 B5 85.7 918.3* 93.3 918.9* 81.5 921.1*
− Cells (1×106 cells/well) were stimulated with SL (1000 mg/ml) at various times after incubation. The amount of NO− 2 or NO2 − plus NO3 released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean9S.E. of three independent experiments each run in duplicate. * PB0.05, significantly different from the control. a
peritoneal macrophages was progressively inhibited with an increasing amount of N GMMA protein. The SL-induced accumulation of nitrite was significantly blocked by N GMMA (0.1 – 10 mM; Fig. 2).
potently inhibited SL induced TNF-a secretion (Table 3). In addition, as Fig. 3 shows, SL-induced use of anti-murine TNF-a neutralizing antibody, indicating that SL-induced TNF-a secretion is crucial for synergistic induction of NO synthesis in mouse peritoneal macrophages.
3.3. Inhibition of SL-induced NO production by STSN To study further the mechanism of synergistic effect of SL on NO synthesis, we examined the influence of PKC inhibitor STSN in rIFN-g plus SL-treated mouse peritoneal macrophages. Adding STSN (20 nM) to the rIFN-g plus SLtreated cells decreased the synergistic effects of SL on NO synthesis (Table 2).
3.4. Effect of SL on rIFN-g-induced TNF-a secretion Mouse peritoneal macrophages secreted very low levels of biologically active TNF-a after 24 h incubation with medium alone, rIFN-g alone, or SL alone (Table 3). However, mouse peritoneal macrophages secreted high levels of biologically active TNF-a after incubation with rIFN-g plus SL. To analyze further the point in the pathway at which PKC is involved in SL-induced signalling, the ability of STSN to block SL-induced bioactive TNF-a secretion was evaluated. STSN NO production was progressively inhibited by the
Fig. 1. Dose-dependent effect of SL on NO synthesis in rIFN-g-treated mouse peritoneal macrophages. Mouse peritoneal macrophages (1 ×106) were cultured with rIFN-g (5 U/ml). The cells were then treated with various concentrations of SL 6 h after incubation. The amount of NO2− released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean 9S.E. of three independent experiments each run in duplicate. *P B 0.05; significantly different from the control.
H.M. Kim et al. / Journal of Ethnopharmacology 67 (1999) 163–169
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Table 3 Effects of SL on rIFN-g, SL, rIFN-g plus SL or rIFN-g plus SL plus STSN-induced TNF-a secretion in mouse peritoneal macrophagesa
Fig. 2. Effect of NO synthase inhibitors on SL-induced NO synthesis in mouse peritoneal macrophages. Mouse peritoneal macrophages (1 × 106) were cultured for 6 h with rIFN-g plus various concentrations of N GMMA. The cells were then treated with SL (1000 mg/ml) and cultured for 42 h. The amount of NO2− released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean 9 S.E. of three independent experiments each run in duplicate. *PB 0.05; significantly different from the control.
4. Discussion In the present study, we demonstrated that SL induces the NO-production in a time and concentration-dependent manner by estimating the accumulated nitrite in the culture medium. NO synthesis in the mouse peritoneal macrophages by Table 2 Effect of STSN on rIFN-g plus SL-induced NO production in mouse peritoneal macrophagesa Addition
STSN
Nitrite concentration (mM)
rIFN-g+SL rIFN-g+SL
− +
45.6 9 3.5 30.89 4.2*
a TG-elicited peritoneal macrophages (1×106 cells/well) were cultured with rIFN-g (5 U/ml) plus SL (1000 mg/ml). The cells were then treated with STSN (20 nM). The amount of NO− 2 released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean9S.E. of three independent experiments each run in duplicate. * PB0.05, significantly different from the control.
Addition rIFN-g
SL
STSN
LPS
TNF-a secretion (ng/ml)
− + + − + +
− − − + + +
− − − − − +
− − + − − −
0.165 90.009 0.455 90.015 3.283 90.153* 0.863 90.082 2.761 90.204* 1.734 90.181
a Mouse peritoneal macrophages (2×105 cells/well) were cultured for 6 h either in medium alone or in medium that contained rIFN-g (5 U/ml) in the absence (−) or presence (+) of STSN (20 nM), and the cells were stimulated with SL (1000 mg/ml). The amount of TNF-a secreted by mouse peritoneal macrophages was measured after 24 h of incubation. Values are the mean9S.E. of three independent experiments each run in duplicate. * PB0.05; significantly different from the control.
SL can be highly stimulated in combination with rIFN-g. SL had a maximal effect on NO synthesis at a concentration of 1000 mg/ml in rIFN-gtreated cells. The results of this study suggest that SL may provide a second signal for synergistic induction of NO synthesis in mouse peritoneal macrophages. NO, the initial product of oxidation of L-arginine, exhibits a multitude of biological actions (Garthwaite et al., 1988; Peunova and Enikolopov, 1995). N GMMA, an analogue of Larginine, inhibited rIFN-g plus SL-induced NO production in mouse peritoneal macrophages. The strong inhibition of nitrite production by N GMMA indicates that it is likely to depend upon a NO synthase. At present, the precise physiological significance of NO synthesis by SL is unknown. However, since NO has emerged as an important intracellular and intercellular regulatory molecule having functions as diverse as vasodilation, neural communication, cell growth regulation and host defense (Moncada et al., 1991), it is tempting to hypothesize that this molecule is involved in the local control of the various fundamental processes. We determined that SL can induce TNF-a secretion in mouse peritoneal macrophages. Probably, this response could appear to reflect a rapid
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but non-specific type of immune activity, in contrast with the slower but highly site-specific recognition actions. In addition, the endogenous mediator NO inhibits the activation of transcription factor NF-kB. Peng et al. (1995) demonstrated that the induction and stabilization of I-kB-a by NO are important mechanism by which NO inhibits NF-kB. Therefore, further work should address the cross-link between SL-induced NO synthesis and the transcription factor NF-kB. It would be interesting to study whether SL would activate NF-kB. In most experimental models, macrophage activation is a two-step process (Russell et al., 1977; Kim and Moon, 1996). The present results demonstrated that the capacity of SL to increase NO synthesis from rIFN-g-primed mouse peritoneal macrophages was the result of SL-induced TNF-a secretion via the signal transduction pathway of PKC activation. The addition of PKC modulator STSN inhibits the synergistic effect of SL with rIFN-g on NO synthesis. Moreover, the addition of STSN inhibited SL-induced TNF-a secretion. Because the induction of NO synthesis by activation signal associated substan-
tially with the release of TNF-a, the inhibition of SL-induced TNF-a secretion illustrates the potential for PKC inhibitor to reduce NO synthesis in mouse peritoneal macrophages. Although the results of this study provide strong evidence that PKC is involved in the NO synthesis, the point in the pathway at which PKC is involved is unknown. In conclusion, our results demonstrate that SL acts as a modulator of mouse peritoneal macrophages activation by rIFN-g via a process involving L-arginine dependent NO production. SL does not appear to function as an activator of NO synthesis by itself. Although the precise mechanism of SL to promote NO synthesis induced by rIFN-g remains to be further elucidated, SL-induced TNF-a secretion by the pathway of PKC is important in the development of macrophage activation.
Acknowledgements This research was supported by Wonkwang University in 1998.
References
Fig. 3. Effect of anti-TNF-a neutralizing antibody on rIFN-g or rIFN-g plus SL-induced NO synthesis in mouse peritoneal macrophages. Mouse peritoneal macrophages (1 × 106) were cultured with rIFN-g (5 U/ml) or rIFN-g plus SL (10 mg/ml), the cells were then treated with anti-TNF-a antibody (dilution, 1:100). The amount of NO2− released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean9 S.E. of three independent experiments each run in duplicate. *PB 0.05; significantly different from the control.
Croen, K.D., 1993. Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type I replication. Journal of Clinical Investigation 91, 2446 – 2452. Garthwaite, J., Charles, S.L., Chess-Williams, R., 1988. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature 336, 385 – 388. Kim, H.M., Moon, Y.H., 1996. Human chorionic gonadotropin induces nitric oxide synthase mRNA in mouse peritoneal macrophages. Biochemical and Biophysical Research Communications 229, 548 – 552. Langerman, J.A.M., Marielle, E.B., Hulst, V.D., Nibbering, P.H., Furth, R.V., 1990. Activation of mouse peritoneal macrophages during infection with salmonella thphimurium does not result in enhanced intracellular killing. Journal of Immunology 144, 4340 – 4346. Lee, E.H., Kim, N.K., Hwang, C.Y., Kim, H.M., 1998. Activation of inducible nitric oxide synthase by YongdamSagan-Tang in mouse peritoneal macrophages. Journal of Ethnopharmacology 60, 61 – 69. Lee, Y.Y., Hsu, F.L., Nohara, T., 1997. Two new soladulcidine glycosides from Solanum and lyratum. Chemical Pharmaceutical Bullutin 45, 1381 – 1382.
H.M. Kim et al. / Journal of Ethnopharmacology 67 (1999) 163–169 Moncada, S., Higgs, A., 1993. The L-arginine-nitric oxide pathway. New England Journal of Medicine 329, 2002– 2012. Moncada, S., Palmer, R.M., Higgs, E.A., 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews 43, 109–142. Narumi, S., Finke, J.H., Hamilton, T.A., 1990. Interferon g and interleukin 2 synergize to induce selective monokine expression in murine peritoneal macrophages. Journal of Biological Chemistry 265, 7036–7041. Nathan, C., 1992. Nitric oxide as a secretory product of mammalian cells. FASEB Journal 6, 3051–3064. Nathan, C., Xie, Q.W., 1994. Regulation of biosynthesis of nitric oxide. Journal of Biological Chemistry 269, 13725– 13728. Peng, H.B., Libby, P., Liao, J.K., 1995. Induction and stabilization of I-kB-a by nitric oxide mediates inhibition of
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NF-kB. Journal of Biological Chemistry 270, 14214 – 14219. Peunova, N., Enikolopov, G., 1995. Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells. Nature 375, 68 – 73. Russell, S.W., Doe, W.F., McIntosh, A.T., 1977. Functional characterization of a stable, noncytolytic stage of macrophage activation in tumors. Journal of Experimental Medicine 146, 1511 – 1520. Scuderi, P., Sterling, R.E., Lam, K.S., Firiley, P.R., Ryan, K.J., Ray, C.G., Petersen, E., Slymen, D.J., Salmon, S.E., 1986. Raised serum levels of tumour necrosis factor in parasitic infections. Lancet 2, 1364 – 1365. Xie, Q.W., Cho, H.J., Calaycay, J., Mumford, R.A., Swiderek, K.M., Lee, T.D., Ding, A., Troso, T., Nathan, C., 1992. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256, 225 – 228.
The nitric oxide-producing properties of Solanum lyratum H.M. Kim a,*, M.J. Kim a, E. Li b, Y.S. Lyu c, C.Y. Hwang c, N.H. An a b
a College of Pharmacy, Wonkwang Uni6ersity, Iksan, Chonbuk 570 -749, South Korea Department of Integration of Traditional and Western Medicine, Hebei Medical Uni6ersity, Hebei, People’s Republic of China c College of Oriental Medicine, Wonkwang Uni6ersity, Iksan, Chonbuk 570 -749, South Korea
Received 27 November 1998; received in revised form 11 January 1999; accepted 18 January 1999
Abstract We examined the effect of Solanum lyratum Thunb. (Solanaceae) (SL) on the production of nitric oxide (NO). Stimulation of mouse peritoneal macrophages with SL after the treatment of recombinant interferon-g (rIFN-g) resulted in increased NO synthesis. SL had no effect on NO synthesis by itself. When SL was used in combination with rIFN-g, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of SL on NO synthesis was shown 6 h after treatment with rIFN-g. The increased production of NO from rIFN-g plus SL-stimulated cells was decreased by the treatment with staurosporin. In addition, synergy between rIFN-g and SL was mainly dependent on SL-induced tumor necrosis factor-a (TNF-a) secretion. All the preparations of SL were endotoxin free. The present results indicate that the capacity of SL to increase NO production from rIFN-g-primed mouse peritoneal macrophages is the result of SL-induced TNF-a secretion via the signal transduction pathway of PKC activation. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Solanum lyratum; Nitric oxide; Mouse peritoneal macrophages; Recombinant interferon-g; Tumor necrosis factor-a
1. Introduction The Solanum lyratum Thunb (Solanaceae) (SL) herbs, well known as ‘Back-Mo-Deung’ in Korea, has been used for regulating body immune function and ability, and it still occupies an important place in traditional Korean medicine. This herb contains solalyrantines A and B, respectively, together with several furostanol, spirostanol and spirosolane glycosides (Lee et al., 1997). Nitric * Corresponding author. Tel.: +82-653-850-6805; fax: + 82-653-843-3421. E-mail address: [email protected] (H.M. Kim)
oxide (NO) is a highly reactive molecule produced from a guanidino nitrogen of L-arginine in a reaction catalyzed by a family of NO synthase (NOS) enzymes (Nathan, 1992). Recent studies have demonstrated the crucial role of NO in the antimicrobial and tumoricidal activities of murine macrophages (Moncada et al., 1991). More recent studies suggest that NO also has antiviral effects in both murine and human cells. Croen (1993) demonstrated that stimulation of a murine macrophage cell line with interferon-gamma (IFN-g) and lipopolysaccharide (LPS) resulted in high level NO production and a 1000-fold inhibition of herpes simplex virus-1 replication. Synthe-
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 1 1 - 2
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H.M. Kim et al. / Journal of Ethnopharmacology 67 (1999) 163–169
sis of high amounts of this reactive radical requires expression of inducible nitric oxide synthase (iNOS), and this initial step is tightly regulated (Nathan and Xie, 1994) in order to prevent potential damage to the host (Moncada and Higgs, 1993). For the production of NO in macrophages, IFN-g is required as a priming signal before those cells can subsequently be triggered by a second signal, for example, LPS, phorbol ester or tumor necrosis factor-a (TNF-a; Nathan, 1992; Lee et al., 1998). In this study, we show that SL synergistically induces NO synthesis by mouse peritoneal macrophages when the cells are treated by recombinant interferon-g (rIFN-g), but SL alone has no significant effect on the NO production. In addition, we will point out that SL has the potency to modulate NO production via the inhibition of protein kinase (PKC) activity, and that synergy between rIFN-g plus SL is mainly dependent on SL-induced TNF-a secretion.
2. Materials and methods
2.1. Reagents Murine rIFN-g (1 ×106 U/mg), recombinant tumor necrosis factor-a (rTNF-a: 1 ×105 U/ml) and rabbit anti-murine TNF-a antibody were purchased from Genzyme (Munchen, Germany). Dulbecco’s modified Eagle’s medium (DMEM), staurosporine (STSN), N-(1-naphthyl)-ethylenediamine dihydrochloride, sodium nitrite, and sulfanilamide were purchased from Sigma (St. Louis, MO). N G-monomethyl-L-arginine (N GMMA) was purchased from Calbiochem (Sandiego, CA). Thioglycollate (TG, fluid thioglycollate medium dehydrated) was purchased from Difco Laboratories (Detroit, MI). All reagents and media for tissue culture experiments were tested for their LPS content with use of a colorimetric Limulus amoebocyte lysate assay (detection limit, 10 pg/ ml; Whittaker Bioproducts, Walkersville, MD). None of these reagents contained endotoxins. Ninety-six-well tissue culture plates and 100-mm diameter petri dishes were purchased from Nunc (Naperville, IL). DMEM containing L-arginine
(84 mg/l), Hank’s balanced salt solution (HBSS), fetal calf serum (FCS), and other tissue culture reagents were purchased from Life Technologies (Grand Island, NY).
2.2. Animals Male C57BL/6 mice were purchased from Dae Han Experimental Animal Center (Eumsung, Korea). The animals were maintained at College of Pharmacy, Wonkwang University. The mice were housed five to ten per cage in a laminar air flow room maintained under a temperature of 229 1°C and relative humidity of 559 10% throughout the study.
2.3. Preparation of extracts The plant sample (Number, 4-96-35) was collected on a farm of Chonnam Province, South Korea. A voucher specimen was deposited at the Herbarium at the College of Pharmacy, Wonkwang University, and extracted with distilled water at 70°C for 5 h. The extract was filtered through a 0.45-mm filter and filtrate was lyophilized. The w/w yield of the extract was about 9.2%. The sample was identified by T.Y. Shin, College of Pharmacy, Woosuk University.
2.4. Cell cultures TG-elicited macrophages were harvested 3 days after i.p. injection of 2.5 ml TG (formula per liter; 15 g of Bacto Casitone, 5 g of Bacto yeast extract, 5.5 g of Bacto dextrose, 2.5 g of sodium chloride, 0.5 g of L-cystine, 0.5 g of sodium thioglycollate, 0.75 g of Bacto agar, 0.001 g of resazurin). We suspended 29.8 g in 1 l distilled water and boiled to dissolve completely and sterilized at 121− 124°C for 15 min and then supplied the prepared medium to mice 8–12 weeks of age, isolated as reported previously (Narumi et al., 1990). Peritoneal lavage was performed by using 8 ml of HBSS, which contained 10 U/ml heparin. Then, the cells were distributed in DMEM, which was supplemented with 10% (v/v) FCS, in either 96well tissue culture plates (2 ×105 cells/well) or 100-mm diameter plastic petri dishes (1×107
H.M. Kim et al. / Journal of Ethnopharmacology 67 (1999) 163–169
cells/dish), incubated for 3 h at 37°C in an atmosphere of 5% CO2, washed three times with HBSS to remove non-adherent cells, and equilibrated with DMEM that contained 10% FCS before treatment.
165
2%-azinobis. OD readings were made within 10 min of addition of the substrate on a Titertek Multiskan with a 405-nm filter. Appropriate specificity controls were included.
2.7. Statistical analysis 2.5. Measurement of nitrite and nitrate 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 supernatants of cultured cells and incubated with an equal volume of the Griess reagent (1% sulfanilamide/0.1% N-(1naphthyl)-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− (nitrite) was determined by using sodium nitrite as a standard. The cellfree medium alone contained 5 – 8 mM of nitrite; this value was determined in each experiment and subtracted from the value obtained from the medium with cells. In some experiments, NO3− (nitrate) was measured by reducing nitrate to nitrite with bacterial nitrate reductase measuring nitrite by using Griess reagent (Langerman et al., 1990).
2.6. Assay of TNF-a secretion TNF-a secretion was measured by modification of an ELISA, as previously described (Scuderi et al., 1986). The ELISA was sensitive to TNF-a concentrations in medium above 40 pg/ml. The ELISA was devised by coating 96-well plates with 6.25 ng/well of murine monoclonal antibody with specificity for human TNF-a. Before use and between subsequent steps in the assay, coated plates were washed twice with PBS containing 0.05% Tween 20 and twice with PBS alone. All reagents used in this assay were incubated for 1 h at room temperature with coated wells. For the standard curve, TNF-a was added to serum previously determined to be negative for endogenous TNF-a. After exposure to medium, assay plates were sequentially exposed to rabbit anti-TNF-a, phosphatase-conjugated goat anti-rabbit IgG, and 2,
Data collected were expressed as means9 S.E. Statistical analysis was performed by the Student’s t-test to express the difference between two groups.
3. Results
3.1. Effect of SL on rIFN-g-induced NO production Initially, we wished to determine whether mouse peritoneal macrophages could be stimulated by SL, either alone or in combination with rIFN-g, to induce NO production. Isolated mouse peritoneal macrophages were cultured either in medium alone or in medium that contained rIFNg (5 U/ml). Then, cells were stimulated with SL at various times during 48 h culture, and NO release was measured by using the Griess method. As Table 1 shows, apparently SL alone did not induce NO synthesis, whereas SL in combination with rIFN-g synergistically increased NO synthesis in cultured peritoneal macrophages. The maximum cooperative effect of SL for NO release were shown 6 h after rIFN-g treatment. The dose-dependent effects of SL in the presence of rIFN-g on NO synthesis were shown in Fig. 1. The synergistic effect was maximal at 1000 mg/ml of SL. Concentrations of less than 10 mg/ml were considerably less effective and, in some cases, ineffective.
3.2. Inhibition of SL-induced NO production by N GMMA, a competiti6e inhibitor of NO synthase To determine if the signaling mechanism in SL-induced NO production is involved in the L-arginine-dependent pathway in mouse peritoneal macrophages, the cells were incubated for 6 h in the presence of rIFN-g plus N GMMA. The production of nitrite by rIFN-g plus SL in mouse
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Table 1 Effect of SL on rIFN-g-induced NO production in mouse peritoneal macrophagesa Addition
Final concentration (mM)
rIFN-g
SL
LPS
NO− 2
− NO− 2 plus NO3
− + + − − + + +
− − − + +(0 h) +(0 h) +(6 h) +(12 h)
− − +(10 ng/ml) − − − − −
B5 13.5 92.8 45.4 99.4* 8.4 90.9 B5 44.3 93.3* 46.2 93.7* 39.5 95.1*
B5 28.3 94.1 86.5 921.6* 15.7 92.3 B5 85.7 918.3* 93.3 918.9* 81.5 921.1*
− Cells (1×106 cells/well) were stimulated with SL (1000 mg/ml) at various times after incubation. The amount of NO− 2 or NO2 − plus NO3 released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean9S.E. of three independent experiments each run in duplicate. * PB0.05, significantly different from the control. a
peritoneal macrophages was progressively inhibited with an increasing amount of N GMMA protein. The SL-induced accumulation of nitrite was significantly blocked by N GMMA (0.1 – 10 mM; Fig. 2).
potently inhibited SL induced TNF-a secretion (Table 3). In addition, as Fig. 3 shows, SL-induced use of anti-murine TNF-a neutralizing antibody, indicating that SL-induced TNF-a secretion is crucial for synergistic induction of NO synthesis in mouse peritoneal macrophages.
3.3. Inhibition of SL-induced NO production by STSN To study further the mechanism of synergistic effect of SL on NO synthesis, we examined the influence of PKC inhibitor STSN in rIFN-g plus SL-treated mouse peritoneal macrophages. Adding STSN (20 nM) to the rIFN-g plus SLtreated cells decreased the synergistic effects of SL on NO synthesis (Table 2).
3.4. Effect of SL on rIFN-g-induced TNF-a secretion Mouse peritoneal macrophages secreted very low levels of biologically active TNF-a after 24 h incubation with medium alone, rIFN-g alone, or SL alone (Table 3). However, mouse peritoneal macrophages secreted high levels of biologically active TNF-a after incubation with rIFN-g plus SL. To analyze further the point in the pathway at which PKC is involved in SL-induced signalling, the ability of STSN to block SL-induced bioactive TNF-a secretion was evaluated. STSN NO production was progressively inhibited by the
Fig. 1. Dose-dependent effect of SL on NO synthesis in rIFN-g-treated mouse peritoneal macrophages. Mouse peritoneal macrophages (1 ×106) were cultured with rIFN-g (5 U/ml). The cells were then treated with various concentrations of SL 6 h after incubation. The amount of NO2− released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean 9S.E. of three independent experiments each run in duplicate. *P B 0.05; significantly different from the control.
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Table 3 Effects of SL on rIFN-g, SL, rIFN-g plus SL or rIFN-g plus SL plus STSN-induced TNF-a secretion in mouse peritoneal macrophagesa
Fig. 2. Effect of NO synthase inhibitors on SL-induced NO synthesis in mouse peritoneal macrophages. Mouse peritoneal macrophages (1 × 106) were cultured for 6 h with rIFN-g plus various concentrations of N GMMA. The cells were then treated with SL (1000 mg/ml) and cultured for 42 h. The amount of NO2− released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean 9 S.E. of three independent experiments each run in duplicate. *PB 0.05; significantly different from the control.
4. Discussion In the present study, we demonstrated that SL induces the NO-production in a time and concentration-dependent manner by estimating the accumulated nitrite in the culture medium. NO synthesis in the mouse peritoneal macrophages by Table 2 Effect of STSN on rIFN-g plus SL-induced NO production in mouse peritoneal macrophagesa Addition
STSN
Nitrite concentration (mM)
rIFN-g+SL rIFN-g+SL
− +
45.6 9 3.5 30.89 4.2*
a TG-elicited peritoneal macrophages (1×106 cells/well) were cultured with rIFN-g (5 U/ml) plus SL (1000 mg/ml). The cells were then treated with STSN (20 nM). The amount of NO− 2 released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean9S.E. of three independent experiments each run in duplicate. * PB0.05, significantly different from the control.
Addition rIFN-g
SL
STSN
LPS
TNF-a secretion (ng/ml)
− + + − + +
− − − + + +
− − − − − +
− − + − − −
0.165 90.009 0.455 90.015 3.283 90.153* 0.863 90.082 2.761 90.204* 1.734 90.181
a Mouse peritoneal macrophages (2×105 cells/well) were cultured for 6 h either in medium alone or in medium that contained rIFN-g (5 U/ml) in the absence (−) or presence (+) of STSN (20 nM), and the cells were stimulated with SL (1000 mg/ml). The amount of TNF-a secreted by mouse peritoneal macrophages was measured after 24 h of incubation. Values are the mean9S.E. of three independent experiments each run in duplicate. * PB0.05; significantly different from the control.
SL can be highly stimulated in combination with rIFN-g. SL had a maximal effect on NO synthesis at a concentration of 1000 mg/ml in rIFN-gtreated cells. The results of this study suggest that SL may provide a second signal for synergistic induction of NO synthesis in mouse peritoneal macrophages. NO, the initial product of oxidation of L-arginine, exhibits a multitude of biological actions (Garthwaite et al., 1988; Peunova and Enikolopov, 1995). N GMMA, an analogue of Larginine, inhibited rIFN-g plus SL-induced NO production in mouse peritoneal macrophages. The strong inhibition of nitrite production by N GMMA indicates that it is likely to depend upon a NO synthase. At present, the precise physiological significance of NO synthesis by SL is unknown. However, since NO has emerged as an important intracellular and intercellular regulatory molecule having functions as diverse as vasodilation, neural communication, cell growth regulation and host defense (Moncada et al., 1991), it is tempting to hypothesize that this molecule is involved in the local control of the various fundamental processes. We determined that SL can induce TNF-a secretion in mouse peritoneal macrophages. Probably, this response could appear to reflect a rapid
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but non-specific type of immune activity, in contrast with the slower but highly site-specific recognition actions. In addition, the endogenous mediator NO inhibits the activation of transcription factor NF-kB. Peng et al. (1995) demonstrated that the induction and stabilization of I-kB-a by NO are important mechanism by which NO inhibits NF-kB. Therefore, further work should address the cross-link between SL-induced NO synthesis and the transcription factor NF-kB. It would be interesting to study whether SL would activate NF-kB. In most experimental models, macrophage activation is a two-step process (Russell et al., 1977; Kim and Moon, 1996). The present results demonstrated that the capacity of SL to increase NO synthesis from rIFN-g-primed mouse peritoneal macrophages was the result of SL-induced TNF-a secretion via the signal transduction pathway of PKC activation. The addition of PKC modulator STSN inhibits the synergistic effect of SL with rIFN-g on NO synthesis. Moreover, the addition of STSN inhibited SL-induced TNF-a secretion. Because the induction of NO synthesis by activation signal associated substan-
tially with the release of TNF-a, the inhibition of SL-induced TNF-a secretion illustrates the potential for PKC inhibitor to reduce NO synthesis in mouse peritoneal macrophages. Although the results of this study provide strong evidence that PKC is involved in the NO synthesis, the point in the pathway at which PKC is involved is unknown. In conclusion, our results demonstrate that SL acts as a modulator of mouse peritoneal macrophages activation by rIFN-g via a process involving L-arginine dependent NO production. SL does not appear to function as an activator of NO synthesis by itself. Although the precise mechanism of SL to promote NO synthesis induced by rIFN-g remains to be further elucidated, SL-induced TNF-a secretion by the pathway of PKC is important in the development of macrophage activation.
Acknowledgements This research was supported by Wonkwang University in 1998.
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Fig. 3. Effect of anti-TNF-a neutralizing antibody on rIFN-g or rIFN-g plus SL-induced NO synthesis in mouse peritoneal macrophages. Mouse peritoneal macrophages (1 × 106) were cultured with rIFN-g (5 U/ml) or rIFN-g plus SL (10 mg/ml), the cells were then treated with anti-TNF-a antibody (dilution, 1:100). The amount of NO2− released by mouse peritoneal macrophages was measured after 48 h of incubation by the Griess method. Values are the mean9 S.E. of three independent experiments each run in duplicate. *PB 0.05; significantly different from the control.
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