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Effect of nitric oxide on phagocytic activity of lipopolysaccharide-induced macrophages: Possible role of exogenous l -arginine
Cell Biology International 31 (2007) 565e569 www.elsevier.com/locate/cellbi
Effect of nitric oxide on phagocytic activity of lipopolysaccharide-induced macrophages: Possible role of exogenous L-arginine Cemil Tu¨mer a,*, Hakkı Murat Bilgin b, Basra Deniz Obay b, Hu¨da Diken b, Mukadder Atmaca b, Mustafa Kelle b a
Department of Physiology, Faculty of Medicine, Mustafa Kemal University, Uur Mumcu Street, 31100 Hatay, Turkey b Department of Physiology, Dicle University Faculty of Medicine, 21280 Diyarbakır, Turkey Received 22 December 2005; revised 31 October 2006; accepted 29 November 2006
Abstract Among the antimicrobial mechanisms associated with macrophages, NO produced by iNOS plays a major role in intracellular killing, but the relationship between NO and phagocytic activity after injection of inflammatory agents into the peritoneal cavity is not clear. The aim of the present study was to investigate the effect of nitric oxide (NO) on macrophage function after treatment with intraperitoneal lipopolysaccharide (LPS) and the role of exogenous L-arginine administration in this event. Six experimental groups and one control group, each consisting of seven Wistar rats were used: Group I: Control; Group II: LPS; Group III: LPS þ L-arginine; Group IV: LPS þ L-arginine þ Aminoguanidine; Group V: LPS þ Aminoguanidine; Group VI: L-arginine; Group VII: Aminoguanidine. Macrophage phagocytic activity and total plasma nitrite levels were increased in the LPS group. In the LPS þ L-arginine group, both the phagocytic activity and total plasma nitrite levels showed large increases. Administration of aminoguanidine (AG), a specific iNOS inhibitor, abolished macrophage phagocytic activity and total plasma nitrite levels in the LPS and LPS þ L-arginine groups. As a result, we showed that NO produced by macrophages has a role not only in intracellular killing, but also in phagocytic activity. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Nitric oxide; Phagocytic activity; L-Arginine; Aminoguanidine; Lipopolysaccharide
1. Introduction Macrophage activation is a key component of the immune response. Several pro-inflammatory cytokines and bacterial products such as lipopolysaccharide (LPS) participate actively in this process (Nathan, 1992; Terenzi et al., 1995; MacMicking et al., 1997). Besides activating macrophages, LPS induces the synthesis of additional cytokines such as tumor necrosis factor a (TNFa), interlekin-1b (IL-1b, and IL-6, which leads to the amplification of the original response (Bone, 1991; Cohen
* Corresponding author. Tel.: þ90 326 214 2636. E-mail address: [email protected] (C. Tu¨mer).
and Glauser, 1991). Activated macrophages release nitric oxide (NO), which is an important bactericidal, anti tumoral and cytotoxic gas (Nathan, 1992; Lorsbach et al., 1993; MacMicking et al., 1997). NO is a product of the conversion of L-arginine to L-citrulline, which is catalyzed by the enzyme NO synthase (NOS) (Michel and Feron, 1997; Ignarro, 2002). Three isoforms of NOS have been cloned and characterized: endothelial NOS, neuronal NOS, and inducible NOS (iNOS) (West et al., 2001). NO that is produced in low levels by the endothelial and neuronal NOS isoforms functions as a signaling molecule in several biological processes, including the regulation of vascular tone and neuronal signaling (Moncada et al., 1991; Schmidt, 1992; Snyder and Bredt, 1992). NO, produced in
1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.11.029
C. Tu¨mer et al. / Cell Biology International 31 (2007) 565e569
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large quantities following induction of iNOS by cytokines and LPS, can have cytotoxic or cytostatic effects on macrophage (Nathan, 1992). iNOS is expressed in various cell types, which include vascular smooth muscle cells, hepatocytes, astrocytes, and macrophages and is induced in response to proinflammatory cytokines or bacterial LPS (Lyons et al., 1992; Schmidt, 1992; Galea et al., 1992; Geller et al., 1993). Since plasma NO released from the cells rapidly auto oxidizes to yield the stable metabolites of NO (nitrite/nitrate), the level of nitrite/nitrate in blood may be an indicator of endogenous NO production (Node et al., 1997). It is widely accepted that NOS activity depends upon the presence of L-arginine and the source of this L-arginine is primarily exogenous (Hrabak and Temesi, 1994). However, the importance of the extracellular L-arginine in vivo is more difficult to quantify (Currie, 1978). Aminoguanidine (AG), an inhibitor that is relatively more selective for iNOS, has been widely used to investigate the role of iNOS in host defense against infections caused by various pathogens (De Groote et al., 1996; Eisenstein et al., 1998). Among the antimicrobial mechanisms associated with the macrophages, NO produced by iNOS, plays a major role in intracellular killing (MacMicking et al., 1997) but the relationship of NO with the phagocytic activity after the injection of inflammatory agents to the peritoneal cavity is not clear. Therefore, in this study, we stimulated the macrophages with LPS in vivo and the phagocytic activity was investigated. The other aim of this study was to investigate the effect of L-arginine on macrophage phagocytic activity stimulated by LPS in vivo. 2. Materials and methods 2.1. Animals In this study, 49 male Wistar Albino rats (200e250 g), obtained from Dicle University Medical Research Center (Diyarbakır, Turkey) were used. All the rats were housed in a 12:12 h dark/light cycle and allowed food and water ad libitum. The committee for animal experiments of the Dicle University Medical Research Center (Diyarbakır, Turkey) gave its approval for the project _ (project number: 30.2.DIC.0.01.00.00/2906).
2.2. Preparation of reagents All chemicals and reagents were purchased from Sigma (Steinheim, Germany). Salmonella typhimurium lipopolysaccharide (LPS), aminoguanidine and L-arginine were dissolved in 0.9% NaCl at concentrations of 4 mg/kg, 50 mg/kg and 10 mg/kg body weight, respectively, and stored at 4 C.
Group V: LPS þ Aminoguanidine group Group VI: L-arginine group Group VII: Aminoguanidine group. The rats were anesthetized with ether 24 h after the injection of these agents.
2.4. Phagocytic activity assays Peritoneal macrophages were obtained by lavage with PBS (pH 7.2e7.4) and suspended in cold (4 C) Hanks’ Balanced Salt Solution (HBSS) with 5% fetal calf serum at an approximate concentration of 2 106 cells/ml. The viability of the macrophage was always >95% as determined by trypan blue exclusion. The suspension of macrophages in HBSS was incubated at 37 C for 30 min in a water bath, with stirring. A suspension of heat killed Saccharomyces cerevisiae yeast cells (40 106 cells/ml) in HBSS was added at an equal volume to the macrophage solution and incubated in a separate tube for 60 min. After the incubation, phagocytic activity was evaluated in terms of phagocytosis number (% phagocytic cell in population, macrophages that had ingested at least one yeast particle) and phagocytosis index (mean number of yeast particles absorbed by one cell) and microscopically enumerated by counting 200 cells (the nonphagocytosing cells too) (Stolka et al., 2001).
2.5. Measurement of plasma levels of total nitrite Since NO is a very labile molecule, its direct measurement in the biological samples is very difficult (Moncada et al., 1991). In an aqueous solution, NO reacts with molecular oxygen and accumulates in the plasma as nitrite and nitrate ions. Therefore, the stable oxidation and products of NO, nitrite (NO 2 ) and nitrate (NO3 ), can be readily measured in biological fluids and have been used in vitro and in vivo as indicators of NO production (Koltuksuz et al., 2000;). Plasma nitrite levels were measured with the Griess reaction (Cortas and Wakid, 1990). Briefly, samples were initially deproteinized with Somogyi reagent (Somogyi, 1930). Total nitrite (nitrite þ nitrate) was measured after the reduction of nitrate to nitrite by copporized cadmium granules (spectrophotometer at 545 nm). A standard curve was established with a set of serial dilutions (108e103 mol/l) of sodium nitrite. Linear regression was performed using the peak area from the nitrite standard. The resulting equation was then used to calculate the unknown sample concentrations. Results were expressed as mmoles per liter of plasma. Therefore, plasma nitrite and nitrate (total nitrite) concentrations were accepted as an index of NO.
2.6. Statistical analysis Results are expressed as the mean S.E. The KruskaleWallis test was used to compare the groups. In two-group comparisons, the ManneWhitney U-test was performed. p < 0.05 was considered statistically significant.
3. Results All animals survived for 24 h after injections. No differences were found among control and experimental groups concerning the total number of macrophages found in rat peritoneal lavage fluid.
2.3. Experimental protocols All agents were administered to the animals intraperitoneally as a single dose 24 h before sacrifice. Each dose was given in 0.5 ml, 0.9% NaCl. The rats were divided into seven groups, each consisting of 7 rats: Group Group Group Group
I: Control Group, animals were injected with 0.5 ml of 0.9% NaCl II: LPS group III: LPS þ L-arginine group IV: LPS þ L-arginine þ Aminoguanidine group
3.1. Effect of LPS and exogenous L-arginine on the macrophage phagocytic activity In LPS group both phagocytosis number and phagocytosis index increased 24 h after LPS injection. Phagocytosis number increased from 68 0.65% to 74 1.41% ( p < 0.05) (Fig. 1) while phagocytosis index increased from 1.85 0.19 to
AG
L-arginine
LPS+AG
LPS+ Larginine+AG
LPS+ Larginine
Fig. 1. The effect of LPS (4 mg/kg), exogenous L-arginine (10 mg/kg) and AG (50 mg/kg) on the phagocytosis number (macrophages that ingested at least one yeast particle). Values are mean S.E.M. ap < 0.05 vs. control and LPS þ AG groups; bp < 0.01 vs. control and LPS þ L-arginine þ AG groups; c p < 0.05 vs. LPS group.
AG
L-arginine
60
LPS
65
a
LPS+AG
70
b,c
LPS+ Larginine+AG
a 75
567
LPS+ Larginine
80
4,5 4 3,5 3 2,5 2 1,5 1 0,5 0
LPS
phagocytic index
b,c
Control
85
Control
phagocytosis number (%phagocytic cell in population)
C. Tu¨mer et al. / Cell Biology International 31 (2007) 565e569
Fig. 2. The effect of LPS (4 mg/kg), exogenous L-arginine (10 mg/kg) and AG (50 mg/kg) on the phagocytosis index (mean number of yeast particles absorbed by one cell). Values are mean S.E.M. ap < 0.05 vs. control and LPS þ AG groups; bp < 0.01 vs. control group; cp < 0.05 vs. LPS and LPS þ L-arginine þ AG groups.
3.4. Effect of AG administration on plasma total nitrite levels
2.8 0.27 ( p < 0.05) (Fig. 2). Exogenous L-arginine supplementation with LPS (LPS þ L-arginine group) resulted with further increases in phagocytosis number from 68 0.65% to 80 1.42% ( p < 0.01) (Fig. 1) and phagocytosis index from 1.85 0.19 to 3.8 0.11 ( p < 0.01) (Fig. 2). Nevertheless, no difference was found between control and L-arginine group (in the absence of LPS stimuli) about the phagocytosis number and phagocytosis index.
As shown in Fig. 3, AG reduced the plasma total nitrite levels in both LPS and LPS þ L-arginine groups. Plasma total nitrite level in the LPS þ AG group decreased from 7.71 0.56 to 5.4 0.57 ( p < 0.05). In the LPS þ L-arginine þ AG group, total plasma nitrite levels decreased from 14.2 1.01 to 5.8 0.50 ( p < 0.01). When AG was injected alone, no significant differences were observed in total plasma nitrite levels in comparison with the control group.
3.2. Effect of AG on phagocytic activity
4. Discussion
AG, an inhibitor with a high specificity for iNOS, reduced phagocytic activity of macrophages activated with LPS and LPS þ L-arginine groups. AG injection with LPS (LPS þ AG group) reduced phagocytosis number from 74 1.41% to 69 0.53% ( p < 0.05) (Fig. 1) and phagocytosis index from 2.8 0.27 to 1.98 0.23 ( p < 0.05) (Fig. 2). Similarly when AG was applied with LPS þ L-arginine (LPS þ L-arginine þ AG group), phagocytosis number decreased from 80 1.42% to 70 0.52% ( p < 0.01) (Fig. 1) and phagocytosis index decreased from 3.8 0.11 to 2.04 0.22 ( p < 0.05) (Fig. 2). No significant differences were observed between AG applied group and the control group in terms of phagocytosis number and phagocytosis index.
The mechanism by which the macrophages kill the pathogens is not fully known. The secretion of the microbicidal digestive enzymes and the reactive oxygen products take roles in that period (Babior, 1984). At recent times, NO and reactive nitrogen products have been shown to play a more active role than the reactive oxygen products at the regulation of the immune system and at the antimicrobial functions of the macrophages for some pathogens (Ding et al., 1988). In the present study, alterations in the macrophage phagocytic activity (phagocytosis number and phagocytosis index) related to NO production were examined. The expression of iNOS gene at macrophages stimulated by LPS were stated at several studies and this gave us a reasonable support to suggest a relationship between the phagocytic activity at LPS stimulated macrophages and NO. The increased macrophage phagocytic activity in addition to the increased NO level was observed in the intraperitoneally LPS-injected group. Thus, our results are in agreement with Stolka et al. (2001) who indicated that treatment of culture media with LPS increased NO-dependent phagocytic activity by the stimulation of macrophages. Kwon et al. (2002) demonstrated that a bacterial polysaccharide stimulated macrophages either in vivo or in vitro and NO levels were increased at both conditions as it was higher in vitro. Furthermore, the phagocytic activity increased in parallel with time. It is well known that NO is produced at several tissues beyond macrophages by stimulation of LPS but
3.3. Effect of LPS and exogenous L-arginine on plasma total nitrite levels As shown in Fig. 3, stimulation of macrophage with LPS increased the levels of total nitrite in comparison with control group (from 5.0 0.72 to 7.71 0.56 mM/l) ( p < 0.05). The effect of LPS on plasma total nitrite level further increased in the presence of exogenous L-arginine (from 5.0 0.72 to 14.2 1.01 m/l) ( p < 0.01). The effect of L-arginine alone on plasma total nitrite levels did not reach the established levels of statistical significance.
C. Tu¨mer et al. / Cell Biology International 31 (2007) 565e569 18 b
16 14 12 10
a
8 6 4
AG
L-arginine
LPS+AG
LPS+ Larginine+AG
LPS+ Larginine
0
LPS
2 Control
plasma total nitrite concentration (micro mol/L)
568
Fig. 3. The effect of LPS (4 mg/kg), exogenous L-arginine (10 mg/kg) and AG (50 mg/kg) on total plasma nitrite concentration. Values are means S.E.M. a p < 0.05 vs. control and LPS þ AG groups; bp < 0.01 vs. control, LPS and LPS þ L-arginine þ AG groups.
the increased NO production is mostly related with macrophages. The main aim of this study was to take attention to differences at phagocytic activity besides the NO levels. Significant increases were shown at macrophage phagocytic activity with LPS stimulation in vivo. It has been postulated that L-arginine plays a vital role in the phagocytic activity, antimicrobial and antitumoral functions of the macrophages both in vivo and in vitro (Potenza et al., 2001; Sosroseno, 2004). According to Potenza et al. (2001), adding low-dose of L-arginine to culture media increases the neutrophil phagocytic activity but adding high doses of L-arginine decreases it. Similarly, Takema et al. (1991) determined that the antitumoral activities of macrophages activated by LPS are related to the concentration of L-arginine and the optimal level must be 0.10e0.15 mM. In agreement with the findings of other results (Takema et al., 1991; Potenza et al., 2001), our study indicates that the exposure of macrophage to 10 mg/kg L-arginine (approximately 0.15 mM) enhanced macrophage phagocytic activity, which was stimulated by LPS, in an additive manner. Moreover, injection of L-arginine intraperitoneally had no effect on macrophage phagocytic activity and total plasma nitrite level in the absence of stimulation. Our findings are also in agreement with Chang et al. (1998) who showed that, when macrophages were not stimulated by LPS, only a very small amount of NO was produced in the presence of L-arginine and in the stimulated macrophage, NO production was significantly increased by L-arginine. Contrary to our findings, Albina et al. (1993) demonstrated that low dose of L-arginine concentration increased the phagocytic activity without LPS stimulation. We suggest that these different results originate from the methodological differentiation. AG is a specific iNOS inhibitor and is used to investigate the role of iNOS in the host’s defenses against the infections of several pathogens (De Groote et al., 1996; Eisenstein et al., 1998). AG inhibits NO production at activated macrophages and prevents NO’s effects on the immune system
(Vazquez-Torres et al., 1996). Vazquez-Torres et al. (1996) determined that macrophages stimulated by LPS had reduced levels of both candidacidal effect and NO quantity when they were treated by AG. Potenza et al. (2001) demonstrated that phagocytic activity that was increased by adding L-arginine was decreased by adding NOS inhibitors to the culture media. In our study, AG decreased phagocytic activity that was increased by LPS and LPS þ L-arginine. This inhibition of AG shows NO’s role in phagocytic activity both in vitro and in vivo. Our findings, which indicated that AG did not constitute a difference in phagocytic activity by itself, are in agreement with the results of Vazquez-Torres et al. (1996) and Potenza et al. (2001). Considering the results of the present study, we can confirm that iNOS-originated NO produced from macrophages has a role, not only in intracellular killing, but also in phagocytic activity. Furthermore, injection of exogenous L-arginine was accompanied by an increase in phagocytic activity.
References Albina JE, Cui S, Mateo RB, Reichner JS. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol 1993;150:5080e5. Babior BM. The respiratory burst of phagocytes. J Clin Invest 1984;73: 599e601. Bone RC. The pathogenesis of sepsis. Ann Intern Med 1991;115:457e69. Chang CI, Liao JC, Kuo L. Arginase modulates nitric oxide macrophages. Am J Physiol 1998;274:H342e8. Cohen J, Glauser MP. Septic shock: treatment. Lancet 1991;338:736e9. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36:1440e3. Currie GA. Activated macrophages kill tumour cells by releasing arginase. Nature 1978;273:758e9. De Groote MA, Testerman T, Xu Y, Stauffer G, Fang FC. Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium. Science 1996;272:414e7. Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol 1988;141:2407e12. Eisenstein TK, Meissler JJ, Miller SI, Stocker BA. Immunosuppression and nitric oxide production induced by parenteral live Salmonella vaccines do not correlate with protective capacity: a phoP: Tn10 mutant does not suppress but does protect. Vaccine 1998;16:24e32. Galea E, Feinstein DL, Reis DJ. Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures. Proc Natl Acad Sci USA 1992;89:10945e9. Geller DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di Silvio M, Wang C, et al. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc Natl Acad Sci 1993;90:3491e5. Hrabak A, Temesi A. Arginine supply fornitric oxide synthesis and arginase is mainly exogenous unelicited murine and rat macrophages. Life Sci 1994; 55:797e805. Ignarro LJ. Nitric oxide as a unique signaling molecule in the vascular system: a historical overview. J Physiol Pharmacol 2002;53:503e14. Koltuksuz U, Irmak MK, Karaman A, Uz E, Var A, Ozyurt H, et al. Testicular nitric oxide levels after unilateral testicular torsion/detorsion in rats pretreated with caffeic acid phenethyl ester. Urol Res 2000;28:360e3. Kwon KH, Kım KI, Jun WJ, Shin DH, Cho HY, Hong BS. In vitro and in vivo Effects of macrophage-stimulatory polysaccharide from leaves of Perilla frutescens var. Crispa. Biol Pharm Bull 2002;25:367e71. Lorsbach RB, Murphy WJ, Lowenstein CJ, Snyder SH, Russell SW. Expression of the nitric oxide synthase gene in mouse macrophages activated
C. Tu¨mer et al. / Cell Biology International 31 (2007) 565e569 for tumor cell killing: molecular basis for the synergy between interferon-g and lipopolysaccharide. J Biol Chem 1993;268:1908e13. Lyons CR, Orloff GJ, Cunningham JM. Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. J Biol Chem. 1992;267:6370e4. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol 1997;15:323e50. Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest 1997;100:2146e52. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109e42. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992;6:3051e64. Node K, Kitakaze M, Yoshikawa H, Kosaka H, Hori M. Reduced plasma concentrations of nitrogen oxide in individuals with essential hypertension. Hypertension 1997;30:405e8. Potenza MA, Nacci C, Mitolo-Chieppa D. Immunoregulatory Effects of Larginine and therapeutical implications. Curr Drug Targets Immune Endocr Metabol Disord 2001;1:67e77. Schmidt HH. NO, CO and OH: endogenous soluble guanylyl cyclase activating factors. FEBS Lett 1992;307:102e7. Snyder SH, Bredt DS. Biological roles of nitric oxide. Sci Am 1992;266:68e71.
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Stolka R, Kashchak N, Kordovski ML, Boyko M, Barska M, Tsyrulnyk A. In vitro respons of phagocytic cells to immunomodulating agents. Med Sci Monit 2001;7:652e8. Somogyi M. A method for the preparation of blood filtrates for the determination of sugar. J Biol Chem 1930;86:655e63. Sosroseno W. The effect of L-arginine on Porphyromonas gingivalis-induced phagocytosis of a murine macrophage-like RAW264.7 cell line. Immunopharmacol Immunotoxicol 2004;26:309e13. Takema M, Inaba K, Uno K, Kakihara K, Tawara K, Muramatsu S. Effect of L-arginine on the retention of macrophage tumoricidal activity. J Immunol 1991;15:1928e33. Terenzi F, Diaz-Guerra MJ, Casado M, Hortelano S, Leoni S, Bosca L. Bacterial lipopeptides induce nitric oxide synthase and promote apoptosis through nitric oxide-independent pathways in rat macrophages. J Biol Chem 1995;270:6017e21. Vazquez-Torres A, Jones-Carson J, Balish E. Peroxynitrite contributes to the candidacidal activity of nitric oxide-producing macrophages. Infect Immun 1996;64:3127e33. West NEJ, Qian HS, Guzik TJ, Black E, Cai S, George SE, et al. Nitric oxide synthase (nNOS) gene transfer modifies venous bypass graft remodeling: effects on vascular smooth muscle cell differentiation and superoxide production. Circulation 2001;104:1526e32.
Effect of nitric oxide on phagocytic activity of lipopolysaccharide-induced macrophages: Possible role of exogenous L-arginine Cemil Tu¨mer a,*, Hakkı Murat Bilgin b, Basra Deniz Obay b, Hu¨da Diken b, Mukadder Atmaca b, Mustafa Kelle b a
Department of Physiology, Faculty of Medicine, Mustafa Kemal University, Uur Mumcu Street, 31100 Hatay, Turkey b Department of Physiology, Dicle University Faculty of Medicine, 21280 Diyarbakır, Turkey Received 22 December 2005; revised 31 October 2006; accepted 29 November 2006
Abstract Among the antimicrobial mechanisms associated with macrophages, NO produced by iNOS plays a major role in intracellular killing, but the relationship between NO and phagocytic activity after injection of inflammatory agents into the peritoneal cavity is not clear. The aim of the present study was to investigate the effect of nitric oxide (NO) on macrophage function after treatment with intraperitoneal lipopolysaccharide (LPS) and the role of exogenous L-arginine administration in this event. Six experimental groups and one control group, each consisting of seven Wistar rats were used: Group I: Control; Group II: LPS; Group III: LPS þ L-arginine; Group IV: LPS þ L-arginine þ Aminoguanidine; Group V: LPS þ Aminoguanidine; Group VI: L-arginine; Group VII: Aminoguanidine. Macrophage phagocytic activity and total plasma nitrite levels were increased in the LPS group. In the LPS þ L-arginine group, both the phagocytic activity and total plasma nitrite levels showed large increases. Administration of aminoguanidine (AG), a specific iNOS inhibitor, abolished macrophage phagocytic activity and total plasma nitrite levels in the LPS and LPS þ L-arginine groups. As a result, we showed that NO produced by macrophages has a role not only in intracellular killing, but also in phagocytic activity. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Nitric oxide; Phagocytic activity; L-Arginine; Aminoguanidine; Lipopolysaccharide
1. Introduction Macrophage activation is a key component of the immune response. Several pro-inflammatory cytokines and bacterial products such as lipopolysaccharide (LPS) participate actively in this process (Nathan, 1992; Terenzi et al., 1995; MacMicking et al., 1997). Besides activating macrophages, LPS induces the synthesis of additional cytokines such as tumor necrosis factor a (TNFa), interlekin-1b (IL-1b, and IL-6, which leads to the amplification of the original response (Bone, 1991; Cohen
* Corresponding author. Tel.: þ90 326 214 2636. E-mail address: [email protected] (C. Tu¨mer).
and Glauser, 1991). Activated macrophages release nitric oxide (NO), which is an important bactericidal, anti tumoral and cytotoxic gas (Nathan, 1992; Lorsbach et al., 1993; MacMicking et al., 1997). NO is a product of the conversion of L-arginine to L-citrulline, which is catalyzed by the enzyme NO synthase (NOS) (Michel and Feron, 1997; Ignarro, 2002). Three isoforms of NOS have been cloned and characterized: endothelial NOS, neuronal NOS, and inducible NOS (iNOS) (West et al., 2001). NO that is produced in low levels by the endothelial and neuronal NOS isoforms functions as a signaling molecule in several biological processes, including the regulation of vascular tone and neuronal signaling (Moncada et al., 1991; Schmidt, 1992; Snyder and Bredt, 1992). NO, produced in
1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.11.029
C. Tu¨mer et al. / Cell Biology International 31 (2007) 565e569
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large quantities following induction of iNOS by cytokines and LPS, can have cytotoxic or cytostatic effects on macrophage (Nathan, 1992). iNOS is expressed in various cell types, which include vascular smooth muscle cells, hepatocytes, astrocytes, and macrophages and is induced in response to proinflammatory cytokines or bacterial LPS (Lyons et al., 1992; Schmidt, 1992; Galea et al., 1992; Geller et al., 1993). Since plasma NO released from the cells rapidly auto oxidizes to yield the stable metabolites of NO (nitrite/nitrate), the level of nitrite/nitrate in blood may be an indicator of endogenous NO production (Node et al., 1997). It is widely accepted that NOS activity depends upon the presence of L-arginine and the source of this L-arginine is primarily exogenous (Hrabak and Temesi, 1994). However, the importance of the extracellular L-arginine in vivo is more difficult to quantify (Currie, 1978). Aminoguanidine (AG), an inhibitor that is relatively more selective for iNOS, has been widely used to investigate the role of iNOS in host defense against infections caused by various pathogens (De Groote et al., 1996; Eisenstein et al., 1998). Among the antimicrobial mechanisms associated with the macrophages, NO produced by iNOS, plays a major role in intracellular killing (MacMicking et al., 1997) but the relationship of NO with the phagocytic activity after the injection of inflammatory agents to the peritoneal cavity is not clear. Therefore, in this study, we stimulated the macrophages with LPS in vivo and the phagocytic activity was investigated. The other aim of this study was to investigate the effect of L-arginine on macrophage phagocytic activity stimulated by LPS in vivo. 2. Materials and methods 2.1. Animals In this study, 49 male Wistar Albino rats (200e250 g), obtained from Dicle University Medical Research Center (Diyarbakır, Turkey) were used. All the rats were housed in a 12:12 h dark/light cycle and allowed food and water ad libitum. The committee for animal experiments of the Dicle University Medical Research Center (Diyarbakır, Turkey) gave its approval for the project _ (project number: 30.2.DIC.0.01.00.00/2906).
2.2. Preparation of reagents All chemicals and reagents were purchased from Sigma (Steinheim, Germany). Salmonella typhimurium lipopolysaccharide (LPS), aminoguanidine and L-arginine were dissolved in 0.9% NaCl at concentrations of 4 mg/kg, 50 mg/kg and 10 mg/kg body weight, respectively, and stored at 4 C.
Group V: LPS þ Aminoguanidine group Group VI: L-arginine group Group VII: Aminoguanidine group. The rats were anesthetized with ether 24 h after the injection of these agents.
2.4. Phagocytic activity assays Peritoneal macrophages were obtained by lavage with PBS (pH 7.2e7.4) and suspended in cold (4 C) Hanks’ Balanced Salt Solution (HBSS) with 5% fetal calf serum at an approximate concentration of 2 106 cells/ml. The viability of the macrophage was always >95% as determined by trypan blue exclusion. The suspension of macrophages in HBSS was incubated at 37 C for 30 min in a water bath, with stirring. A suspension of heat killed Saccharomyces cerevisiae yeast cells (40 106 cells/ml) in HBSS was added at an equal volume to the macrophage solution and incubated in a separate tube for 60 min. After the incubation, phagocytic activity was evaluated in terms of phagocytosis number (% phagocytic cell in population, macrophages that had ingested at least one yeast particle) and phagocytosis index (mean number of yeast particles absorbed by one cell) and microscopically enumerated by counting 200 cells (the nonphagocytosing cells too) (Stolka et al., 2001).
2.5. Measurement of plasma levels of total nitrite Since NO is a very labile molecule, its direct measurement in the biological samples is very difficult (Moncada et al., 1991). In an aqueous solution, NO reacts with molecular oxygen and accumulates in the plasma as nitrite and nitrate ions. Therefore, the stable oxidation and products of NO, nitrite (NO 2 ) and nitrate (NO3 ), can be readily measured in biological fluids and have been used in vitro and in vivo as indicators of NO production (Koltuksuz et al., 2000;). Plasma nitrite levels were measured with the Griess reaction (Cortas and Wakid, 1990). Briefly, samples were initially deproteinized with Somogyi reagent (Somogyi, 1930). Total nitrite (nitrite þ nitrate) was measured after the reduction of nitrate to nitrite by copporized cadmium granules (spectrophotometer at 545 nm). A standard curve was established with a set of serial dilutions (108e103 mol/l) of sodium nitrite. Linear regression was performed using the peak area from the nitrite standard. The resulting equation was then used to calculate the unknown sample concentrations. Results were expressed as mmoles per liter of plasma. Therefore, plasma nitrite and nitrate (total nitrite) concentrations were accepted as an index of NO.
2.6. Statistical analysis Results are expressed as the mean S.E. The KruskaleWallis test was used to compare the groups. In two-group comparisons, the ManneWhitney U-test was performed. p < 0.05 was considered statistically significant.
3. Results All animals survived for 24 h after injections. No differences were found among control and experimental groups concerning the total number of macrophages found in rat peritoneal lavage fluid.
2.3. Experimental protocols All agents were administered to the animals intraperitoneally as a single dose 24 h before sacrifice. Each dose was given in 0.5 ml, 0.9% NaCl. The rats were divided into seven groups, each consisting of 7 rats: Group Group Group Group
I: Control Group, animals were injected with 0.5 ml of 0.9% NaCl II: LPS group III: LPS þ L-arginine group IV: LPS þ L-arginine þ Aminoguanidine group
3.1. Effect of LPS and exogenous L-arginine on the macrophage phagocytic activity In LPS group both phagocytosis number and phagocytosis index increased 24 h after LPS injection. Phagocytosis number increased from 68 0.65% to 74 1.41% ( p < 0.05) (Fig. 1) while phagocytosis index increased from 1.85 0.19 to
AG
L-arginine
LPS+AG
LPS+ Larginine+AG
LPS+ Larginine
Fig. 1. The effect of LPS (4 mg/kg), exogenous L-arginine (10 mg/kg) and AG (50 mg/kg) on the phagocytosis number (macrophages that ingested at least one yeast particle). Values are mean S.E.M. ap < 0.05 vs. control and LPS þ AG groups; bp < 0.01 vs. control and LPS þ L-arginine þ AG groups; c p < 0.05 vs. LPS group.
AG
L-arginine
60
LPS
65
a
LPS+AG
70
b,c
LPS+ Larginine+AG
a 75
567
LPS+ Larginine
80
4,5 4 3,5 3 2,5 2 1,5 1 0,5 0
LPS
phagocytic index
b,c
Control
85
Control
phagocytosis number (%phagocytic cell in population)
C. Tu¨mer et al. / Cell Biology International 31 (2007) 565e569
Fig. 2. The effect of LPS (4 mg/kg), exogenous L-arginine (10 mg/kg) and AG (50 mg/kg) on the phagocytosis index (mean number of yeast particles absorbed by one cell). Values are mean S.E.M. ap < 0.05 vs. control and LPS þ AG groups; bp < 0.01 vs. control group; cp < 0.05 vs. LPS and LPS þ L-arginine þ AG groups.
3.4. Effect of AG administration on plasma total nitrite levels
2.8 0.27 ( p < 0.05) (Fig. 2). Exogenous L-arginine supplementation with LPS (LPS þ L-arginine group) resulted with further increases in phagocytosis number from 68 0.65% to 80 1.42% ( p < 0.01) (Fig. 1) and phagocytosis index from 1.85 0.19 to 3.8 0.11 ( p < 0.01) (Fig. 2). Nevertheless, no difference was found between control and L-arginine group (in the absence of LPS stimuli) about the phagocytosis number and phagocytosis index.
As shown in Fig. 3, AG reduced the plasma total nitrite levels in both LPS and LPS þ L-arginine groups. Plasma total nitrite level in the LPS þ AG group decreased from 7.71 0.56 to 5.4 0.57 ( p < 0.05). In the LPS þ L-arginine þ AG group, total plasma nitrite levels decreased from 14.2 1.01 to 5.8 0.50 ( p < 0.01). When AG was injected alone, no significant differences were observed in total plasma nitrite levels in comparison with the control group.
3.2. Effect of AG on phagocytic activity
4. Discussion
AG, an inhibitor with a high specificity for iNOS, reduced phagocytic activity of macrophages activated with LPS and LPS þ L-arginine groups. AG injection with LPS (LPS þ AG group) reduced phagocytosis number from 74 1.41% to 69 0.53% ( p < 0.05) (Fig. 1) and phagocytosis index from 2.8 0.27 to 1.98 0.23 ( p < 0.05) (Fig. 2). Similarly when AG was applied with LPS þ L-arginine (LPS þ L-arginine þ AG group), phagocytosis number decreased from 80 1.42% to 70 0.52% ( p < 0.01) (Fig. 1) and phagocytosis index decreased from 3.8 0.11 to 2.04 0.22 ( p < 0.05) (Fig. 2). No significant differences were observed between AG applied group and the control group in terms of phagocytosis number and phagocytosis index.
The mechanism by which the macrophages kill the pathogens is not fully known. The secretion of the microbicidal digestive enzymes and the reactive oxygen products take roles in that period (Babior, 1984). At recent times, NO and reactive nitrogen products have been shown to play a more active role than the reactive oxygen products at the regulation of the immune system and at the antimicrobial functions of the macrophages for some pathogens (Ding et al., 1988). In the present study, alterations in the macrophage phagocytic activity (phagocytosis number and phagocytosis index) related to NO production were examined. The expression of iNOS gene at macrophages stimulated by LPS were stated at several studies and this gave us a reasonable support to suggest a relationship between the phagocytic activity at LPS stimulated macrophages and NO. The increased macrophage phagocytic activity in addition to the increased NO level was observed in the intraperitoneally LPS-injected group. Thus, our results are in agreement with Stolka et al. (2001) who indicated that treatment of culture media with LPS increased NO-dependent phagocytic activity by the stimulation of macrophages. Kwon et al. (2002) demonstrated that a bacterial polysaccharide stimulated macrophages either in vivo or in vitro and NO levels were increased at both conditions as it was higher in vitro. Furthermore, the phagocytic activity increased in parallel with time. It is well known that NO is produced at several tissues beyond macrophages by stimulation of LPS but
3.3. Effect of LPS and exogenous L-arginine on plasma total nitrite levels As shown in Fig. 3, stimulation of macrophage with LPS increased the levels of total nitrite in comparison with control group (from 5.0 0.72 to 7.71 0.56 mM/l) ( p < 0.05). The effect of LPS on plasma total nitrite level further increased in the presence of exogenous L-arginine (from 5.0 0.72 to 14.2 1.01 m/l) ( p < 0.01). The effect of L-arginine alone on plasma total nitrite levels did not reach the established levels of statistical significance.
C. Tu¨mer et al. / Cell Biology International 31 (2007) 565e569 18 b
16 14 12 10
a
8 6 4
AG
L-arginine
LPS+AG
LPS+ Larginine+AG
LPS+ Larginine
0
LPS
2 Control
plasma total nitrite concentration (micro mol/L)
568
Fig. 3. The effect of LPS (4 mg/kg), exogenous L-arginine (10 mg/kg) and AG (50 mg/kg) on total plasma nitrite concentration. Values are means S.E.M. a p < 0.05 vs. control and LPS þ AG groups; bp < 0.01 vs. control, LPS and LPS þ L-arginine þ AG groups.
the increased NO production is mostly related with macrophages. The main aim of this study was to take attention to differences at phagocytic activity besides the NO levels. Significant increases were shown at macrophage phagocytic activity with LPS stimulation in vivo. It has been postulated that L-arginine plays a vital role in the phagocytic activity, antimicrobial and antitumoral functions of the macrophages both in vivo and in vitro (Potenza et al., 2001; Sosroseno, 2004). According to Potenza et al. (2001), adding low-dose of L-arginine to culture media increases the neutrophil phagocytic activity but adding high doses of L-arginine decreases it. Similarly, Takema et al. (1991) determined that the antitumoral activities of macrophages activated by LPS are related to the concentration of L-arginine and the optimal level must be 0.10e0.15 mM. In agreement with the findings of other results (Takema et al., 1991; Potenza et al., 2001), our study indicates that the exposure of macrophage to 10 mg/kg L-arginine (approximately 0.15 mM) enhanced macrophage phagocytic activity, which was stimulated by LPS, in an additive manner. Moreover, injection of L-arginine intraperitoneally had no effect on macrophage phagocytic activity and total plasma nitrite level in the absence of stimulation. Our findings are also in agreement with Chang et al. (1998) who showed that, when macrophages were not stimulated by LPS, only a very small amount of NO was produced in the presence of L-arginine and in the stimulated macrophage, NO production was significantly increased by L-arginine. Contrary to our findings, Albina et al. (1993) demonstrated that low dose of L-arginine concentration increased the phagocytic activity without LPS stimulation. We suggest that these different results originate from the methodological differentiation. AG is a specific iNOS inhibitor and is used to investigate the role of iNOS in the host’s defenses against the infections of several pathogens (De Groote et al., 1996; Eisenstein et al., 1998). AG inhibits NO production at activated macrophages and prevents NO’s effects on the immune system
(Vazquez-Torres et al., 1996). Vazquez-Torres et al. (1996) determined that macrophages stimulated by LPS had reduced levels of both candidacidal effect and NO quantity when they were treated by AG. Potenza et al. (2001) demonstrated that phagocytic activity that was increased by adding L-arginine was decreased by adding NOS inhibitors to the culture media. In our study, AG decreased phagocytic activity that was increased by LPS and LPS þ L-arginine. This inhibition of AG shows NO’s role in phagocytic activity both in vitro and in vivo. Our findings, which indicated that AG did not constitute a difference in phagocytic activity by itself, are in agreement with the results of Vazquez-Torres et al. (1996) and Potenza et al. (2001). Considering the results of the present study, we can confirm that iNOS-originated NO produced from macrophages has a role, not only in intracellular killing, but also in phagocytic activity. Furthermore, injection of exogenous L-arginine was accompanied by an increase in phagocytic activity.
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