Eur. J. Immunol. 1992. 22: 441-446

Yun Li.0, Alison Severn, Mark V. Rogers, Richard M. J. Palmer, Salvador Moncada and FooY. Liew

The Wellcome Research Laboratories, Beckenham

Catalase inhibits nitric oxide synthesis 441

Catalase inhibits nitric oxide synthesis and the killing of intracellular Leishmania major in murine macrophages

Mouse peritoneal macrophages activated with interferon-y (IFN-y) and lipopo- lysaccharide produce substantial amounts of nitric oxide (NO), which correlates with the elimination of the intracellular protozoan parasite Leishmania major. Both the production of NO and the leishmanicidal function of the activated macrophages can be significantly inhibited by catalase in a dose- and tirne- dependent manner. These results could not be interpreted by the reduction of H202 by catalase since the removal of H202 by the addition of glutathione peroxidase had no effect on the NO synthesis or the leishmanicidal function of activated macrophages. Furthermore, catalase did not affect the induction of NO synthase in IFN-y-activated rnacrophages. In contrast, the inhibition of NO synthesis and leishmanicidal activity by catalase was reversed in a dose-dependent manner by the addition of tetrahydrobiopterin, a cofactor of N O synthase.Taken together, these results not only further support the central role of NO as the cytotoxic moiety, but also suggest that hydrogen peroxide may interfere with NO production by affecting the levels of cofactor needed for its synthesis.

1 Introduction

Currently, there is much interest in the biological role of nitric oxide (NO) for various functions in different cells [I]. NO is the transduction mechanism for the soluble guany- late cyclase responsible for endothelium-dependent vascu- lar relaxation, modulation of platelet reactivity and some forms of central and peripheral neurotransmission [2]. Furthermore, NO contributes to the cytotoxic and antimi- crobial actions of macrophages activated by various immu- nological stimuli [3].

Nitric oxide is derived from molecular oxygen [4] and the guanidino nitrogen of L-arginine [S-71. The reaction is catalyzed by the cytosolic enzyme NO synthase, of which there are at least two distinct types. One is constitutive in neuronal tissue and endothelium and is Ca’+/calmodulin dependent. The other, which is induced by IFN-y, TNF-a and LPS in endothelium, neutrophils, hepatocytes and macrophages, is Ca2+ independent. Both enzymes are dependent on a number of cofactors, including NADPH, FAD and tetrahydrobiopterin (HdB) [8-101. The activity of these enzymes can be specifically and stoichiometrically inhibited by L-arginine structural analogues such as L- NG-mono-methyl arginine (L-NMMA) [11, 121.

[I 98781

0 Supported by a scholarship from the British Council. A Department of Surgery, University of Aberdeen, Aberdeen,

Scotland

Correspondence: Foo Y. Liew, Department of Immunology, University of Glasgow, Glasgow G11 6NT, Scotland

Abbreviations: L-NMMA: L-NG-mono-methylarginine GPX: Glutathione peroxidase H a : Tetrahydrobiopterin SOD: Super- oxide dismutase

Macrophages activated with JFN-y and LPS have been shown to have a powerful cytostatic effect on the fungal pathogen Cryptococcus neofonnans [ 131 and the protozoan Toxoplasma gondii [ 141, the microbiostatic effect being dependent on L-arginine, and inhibited by the presence of L-NMMA. Macrophages similarly activated can also kill the extracellular helminth Schistosoma mansoni in vitro [I51 and the intracellular protozoa Leishmania by means of NO in vitro [16-181 and in vivo [17]. The leishmanicidal function of activated macrophages can be completely inhibited by L-NMMA in a dose-dependent manner. This, together with the earlier finding [19] that L. major can be eliminated by a murine macrophage cell line, IC-21, which is deficient in the production of oxygen metabolites, suggests that NO may be sufficient to account for the leishmanicidal activity of macrophages. However, numer- ous earlier studies have demonstrated that reactive oxygen intermediates (ROI) can kill intracellular and extracellular Leishmania [20-231. Since NO and ROI are synthesized through distinct pathways [24], we examined the relative role of N O and ROI in this macrophage antimicrobial system by modulating the levels of HzO2 with catalase, which converts H202 to molecular oxygen. We report here that catalase inhibits macrophage leishmanicidal activity and this correlates with a reduction in NO synthesis. In addition, the inhibitory effect of catalase can be reversed by the addition of H4B. These results suggest that ROI may interfere with the production of NO and, therefore, with the related cytotoxicity of activated macrophages.

2 Materials and methods

2.1 Mice and parasites

CBA/T6T6 mice aged 8 to 10 weeks were obtained from the colonies at Wellcome Research Laboratories, Beckenham, Kent. The isolation, cultivation and maintenance of the promastigote stage of the parasite L. major (LV39) have been described in detail [25].

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992 0014-2980/92/0202-0441$3.50 + .25/0

442 Y, Li, A. Severn, M.V. Rogers et al. Eur. J. Immunol. 1992. 22: 441-446

2.2 Materials

L-NMMA was generously provided by Dr. H. Hodson of the Department of Medicinal Chemistry, Wellcome Re- search Laboratories, Beckenham, Kent. Murine recombi- nant IFN-y and TNF-a were kindly provided by Dr. G. Ad- olf, Ernst Boehringer-Institute fur Arzneimittel-For- schung, Vienna, Austria. LPS was obtained from Sigma (St. Louis, MO) and [3H]dThd (26 Ci/nmol) was obtained from Amersham International (Amersham, GB). Superox- ide dismutase (SOD) and glutathione peroxidase (GPX) from bovine erythrocytes were obtained from Sigma and bovine liver catalase was purchased from Sigma and Boehringer Mannheim, Mannheim, FRG; catalase stabil- izers thymol and alkyldimethylbenzylammonium chloride were obtained form Sigma. H4B was obtained from Dr. B. Schircks (Jona, Switzerland).

2.3 Leishmanicidal assay

This assay has been described in detail elsewhere [26]. Briefly, PEC were collected in culture medium (RPMI 1640 plus 10% FCS, L-glutamine, penicillin and streptomycin) from CBA mice injected i.p. 4-6 days previously with 3 ml of a 2% sterile, hydrolyzed starch solution (BDH Chemi- cals, Poole, GB). The cells were plated at 1 X lo5 celldo. 1 ml culture mediudwell in 96-well flat-bottom plates (Nunc, Roskilde, Denmark) and were incubated at 37°C in an atmosphere of 5% CO2 in air for 24 h. Nonadherent cells were removed and the adherent cells washed three times with a prewarmed medium.To each well was added 100 pl of medium containing 10 ng/ml of LPS with or without IFN-y (400U/ml). In some cultures, catalase, GPX, SOD, H4B or L-NMMA were also added. Cultures were then incubated as above for 2 4 h before addition of 1 x lo5 L. major promastigotes in 100 pl of culture medium containing 10 ng/ml of LPS to each well. After culture for an additional 72 h, 50 pl of supernatant were removed from each well and stored at - 20 "C for the analysis of NO, content and the cultures were washed extensively with a prewarmed medium. To each well was added 100 pl of 0.01% SDS solution in serum-free medium at 37 "C for 20-30 min. Schneider's medium supplemented with 30% FCS and L-glutamine was added (100 pl/well) and the cultures were incubated at 28 "C for an additional 72 h. The cultures, in three to six replicates, were then pulsed with 1 pCi/well (= 37 kBq) of [3H]dThd and the incorpo- ration of radioactivity by viable parasites after 18 h of further culturing was determined by harvesting the para- sites and counting in a p-counter (p-plate, LKB, Bromma, Sweden). The results were expressed as mean cpm k SEM or as percent killing compared to controls.

2.4 Measurement of NO;

NO, in the culture supernatants was assayed by chemilu- minescence as described previously [27]. This measurement reflects the level of NO produced by the cells [8].

2.5 Measurement of H202 production

H 2 0 2 in the culture supernatants was assayed by a colori- metric method as described by Pick and Keisari [28].

Peritoneal cells were dispensed into a 24-well (Costar, Cambridge, MA) plate at 2 X lo6 celldm1 in culture medium (phenol red-free RPMI 1640 containing 10% FCS). The plate was incubated at 37 "C and 5% COz for 24 h. Nonadherent cells were removed by washing with prewarmed phenol red-free balanced salt solution. The macrophages were then treated with various concentrations of reagents as indicated in Fig. 5 for 48 h at 37 "C and 5% C02. At the end of the culture, cells were washed with prewarmed balanced salt solution and 1 ml of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buf- fer, pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red, and 19 U/ml of horseradish peroxidase and 10 pg/ml of PMA) was added to each well, and incubated for another hour. The cell-free supernatants were transferred to conical glass tubes and centrifuged for 5 min at 2000 x g at 4 "C. The supernatants were then adjusted to pH 12 by addition of 10 $1 N NaOH and absorbance determined at 600 nm.The results were expressed as pmol H20212.5 x lo6 cells/60 min by reference to a standard curve.

2.6 Assay for NO synthase activity

Macrophages in culture medium were distributed into 12-well plates (Costar) at 5 x lo6 cells/well and incubated for 6 h at 37 "C and in an atmosphere of 5% C02 in air. Nonadherent cells were then removed by washing and various concentrations of reagents were added. The cul- tures (2 ml/well) were incubated for 24 h at 37 "C and 5% COz. Supernatants were aspirated and 250 pl of 0.1 M Hepes (pH 7.4) with 1 mM dithiothreitol (Sigma) was added to each well and the cells harvested with disposable rubber policemen and subjected to three cycles of freeze- thawing. The samples were then centrifuged at 100 000 x g for 30 min at 4 "C and the supernatants assayed for NO synthase activity by a spectrophotometric method as de- scribed previously [29]. Briefly, NO synthesis was measured by incubating 0.5-ml aliquots containing 5 p~ oxyhemoglo- bin and 20% (v/v) macrophage cytosol in 40 m M potassium phosphate buffer (pH 7.2) containing MgC12 (1 mM) in a dual wavelength spectrophotometer [Shimadzu (Kyoto, Japan) UV-30001 using a band width of 2 nm, at 37 "C. NO synthesis was initiated by addition of L-arginine (100 p ~ ) and NADPH (100 p ~ ) . The protein content of the cytosol was determined by Coomassie blue dye binding method according to the manufacturer's recommendations (Pierce Chemicals, Rockford, IL).

2.7 Statistical analysis

Results are expressed mean k SEM and statistical signifi- cance 0, < 0.05) was analyzed by Student's t-test.

3 Results

3.1 Effects of catalase on NO synthesis and leishmanicidal function of activated macrophages

3.1.1 Dose-response

When macrophages were activated with IFN-y and LPS, they produced substantial amounts of NO, as measured by

Eur. J. Immunol. 1992. 22: 441-446 Catalase inhibits nitric oxide synthesis 443

production and leishmanicidal function by catalase became less pronounced when the enzyme was added after the macrophages were activated with IFN-y (Fig. 2). However, a significant effect was still evident when the enzymes were added 48 h after IFN-y activation and the supernatants harvested 24 h later.

the levels of NO, in the culture supernatants compared to unactivated macrophages. The synthesis of NO was abol- ished by 800 U/ml of catalase (Fig. 1). Interestingly, a lower concentration (8 U/ml) of this preparation of catalase enhanced the NO synthesis. This enhancing effect, how- ever, was not found in catalase preparations of higher purity and thus may be attributable to contaminants (Sect. 3.2). The production of NO by macrophages is related to their leishmanicidal activity. Thus, the killing of the intracellular parasites by activated macrophages was inhibited by 800 U/ml of catalase but enhanced by 8 U/ml of this batch of enzyme (Fig. 1). In some experiments, the survival of the parasites in activated macrophages cultured with 800 U/ml of catalase was significantly higher than those in control cultures without IFN-y and catalase, indicating that catalase may have inhibited the endogenous production of NO by macrophages cultured with LPS alone, which was added in the medium.

The leishmanicidal activity was routinely estimated by the decrease in the ability of the surviving parasites to incor- porate [3H]dThd, because this method offers a more convenient and objective assay system. Experiments were also carried out by microscopic examination of the macro- phage monolayer at 2, 24, 48 and 72 h after infection, according to a method described previously [26]. IFN-y- activated macrophages were efficient in eliminating the intracellular parasites, with significant killing starting 24 h after infection and no viable parasites detectable 72 h after infection. Catalase reversed this killing, and at 72 h signif- icantly more parasites were present in the catalase-treated macrophages than in the control cultures (Table 1). At the concentrations used, catalase did not affect the viability of the macrophages throughout the incubation period. It also did not affect the parasite uptake by the macrophages or the percentage of infected macrophage 2 h after the infection (data not shown).

3.1.2 Time-course study

The effect of catalase (800 U/ml) on the macrophage functions was time dependent. The inhibition of NO

Table 1. Effect of catalase on the leishmanicidal activity of activated macrophages”)

Macrophages No. parasited100 macrophages cultured with

2 h 24h 48 h 72 h

medium alone 321(21) 301(12) 298(15) 292(17)

IFN-y + catalase IFN-y 330(19) 175(10) -

800 U/ml 315( 15) 298( 11) 320( 14) 410( 12) 80 U/ml 310(17) 322(18) 167(12) 76(11) 8 U/ml 279(20) 156(11) 4OJJ -

a) For details see Sect. 2.3. IFN-y was used at 400 U/ml and all medium contained 10 nglml LPS. Catalase was Lot C-10, (Sigma) sp. act., 2-5000 U/mg. Figures represent mean (k 1 SEM), n = 4. Those underlined are significantly different (p < 0.05) from their respective controls (medium alone).

600

YI - 5 500

4 300 F 0 3

0 200 n

B I !$ 100

f

A

60 a E

50 p 0

X

2

40 t- d 2 8 30 a z

20 E 0

Y

F 10

I - - I A

I ‘ 0 8 80 800

0

CATALASE (UN ITS/ml)

Figure 1 . Effects of catalase on NO synthesis (open symbols) and leishmanicidal function (closed symbols) of macrophages activated by IFN-y and LPS. Control cultures were macrophages either unactivated (V, W), or activated by IFN-y and LPS alone (A, +). Vertical bars = 1 SEM, n = 3-6. The results are representative of three experiments. Catalase was Lot C-10 (Sigma) with sp. act. of 2-5000 U/mg protein.

500

7+ - 400 - 8

?? ‘I

300 - z t- 0 0

2 200 2 a IN

100

3 P

0 24 48 72 TIME (h)

Figure 2. Effects of 800 U/ml of catalase on NO synthesis (open symbols) and leishmanicidal function (closed symbols), added at various times after the activation of macrophages by IFN-y and LPS. Control cultures were either unactivated macrophages (V, B), or macrophages activated with IFN-y and LPS alone (A,. +). Vertical bars = 1 SEM, n = 3 4 . The results are representative of three experiments.

444

3.2 The inhibitory effect of catalase is correlated with

Y. Li, A. Severn, M.V. Rogers et a1

the enzymatic activity

To rule out the possibility that the inhibitory effect of catalase was due to a nonspecific action of contaminants present in the enzyme preparation, we tested the inhibitory effect of three different batches of catalase with specific activities ranging from 2000 to 50000 U/mg protein. All three preparations had identical inhibitory activity based on unit enzyme activity (Fig. 3). The preparation with the lowest specific activity also induced a modest, but signifi- cant, enhancement of NO synthesis, suggesting that this enhancement may be due to contaminants (see also Sect. 3.1 and Fig. 1). The catalase stabilizers, thymol and alkyldimethylbenzylammonium chloride had no effect on the macrophage functions at the concentrations used (Fig. 3). Furthermore, heating at 100 "C for half an hour completely abolished the inhibitory activity of catalase (data not shown).

Eur. J. Immunol. 1992. 22: 441-446

Table2. Effects of catalase and GPX on the induction of NO synthase and NO production by activated macrophages")

3.3 GPX has no effect on NO synthesis or leishmanicidal activity

To investigate whether the inhibitory effect of catalase was due to the removal of H202,we tested the effect of GPX on the NO synthesis and leishmanicidal function of activated macrophages. IFN-y-activated macrophages are highly leishmanicidal and produced substantial levels of HzO2 and NO (Fig. 4). Furthermore, the leishmanicidal activity and the NO synthesis, but not the production of H202, were inhibited by L-NMMA. In contrast, GPX, though remov- ing completely H202, had no effect on NO synthesis or leishmanicidal activities, both of which were inhibited by L-NMMA .

300

C 0 .- c 4 100 e a

0

I , ,

" 8 80 800 Catalase (u/rnl)

Figure 3. Effects of different batches of catalase on NO synthesis by macrophages activated with IFN-y and LPS. Bovine liver catalase from Sigma (Lot C-10, sp. act. 2-5000 Ulmg protein 0; and Lot C-3155, sp. act. 50,000 U/mg, A), and from Boehringer Mannheim (Lot 106810, sp. act. 65,000 U/mg, W) had indistingui- shable inhibitory effects on the basis of unit enzyme activity. Control cultures did not include catalase (0) or include the equivalent amounts of catalase stabilizers: thymol (A) or alkyldi- methylbenzylammonium chloride (0). Asterisk denotes signifi- cant difference (p < 0.05) from control (0).

Treatment of NO synthase activity NO, macrophages (pmollmg proteinlmin) (pmoI1100 cells)

IFN-y 29.3 It 3.0 450 rt 5 IFN-y + catalase 32.5 f 4.2 55.6 f 14 IFN-y + GPX 34.1 f 4.5 475.Y f 64

a) Macrophages were activated with IFN-y (400 U/ml) and LPS (10nglml) in the presence of catalase (800U/ml), or GPX (10 units/ml). NO synthase activity and NO, production by the macrophages were assayed as described in Sect. 3. Figures are mean f 1 SEM, n = 3.The results are representative of three experiments. Figure underlined is significantly different 0, < 0.01) from control (IFN-y).

6ool 400L w -

0

p 200

O4 0 t f 2o "I

+ Figure 4. Effects of glutathione peroxidase (GPX, 10 units/ml), and L-NMMA ( 5 0 ~ ~ ) on macrophages activated with IFN-y (400 U/ml) and LPS (10 ng/ml) in terms of leishmanicidal activity, H202 production (2.5 pM X lo6 cells/60 min), and NO synthesis (pmol/105 cells/72 h).

Eur. J. Immunol. 1992. 22: 441-446 Catalase inhibits nitric oxide synthcsis 445

3.4 Effects of catalase and GPX on the induction of NO synthase in activated macrophages

We also investigated the effect of catalase and GPX on the induction of NO synthase in activated macrophages. Although catalase strongly inhibited N O synthesis, it had no significant effect on the induction of NO synthase by IFN-y in these cells (Table 2). GPX, on the other hand, had no effect on NO synthesis or the induction of NO synthase.

3.5 The inhibitory effect of catalase can be reversed by H4B

We tested the possibility that the activity of catalase may have resulted in the oxidization of the cofactor H4B, rendering it unavailable for the synthesis of NO. H4B was added to the cultures of IFN-y-activated macrophages in the presence of catalase. As above, the production of NO and leishmanicidal function of activated macrophages were significantly inhibited by catalase, but these were progres- sively reversed by increasing concentrations of H4B and returned to the control levels with SO0 VM of H4B (Fig. 5). H4B alone had no effect on the synthesis of NO or the leishmanicidal function of the activated macrophages (data not shown).

1200 ,-. vt - - d 1000

ro 0 - 2 800 E v 600 0 .- +-

400 e Q

I N 200 0 z

0

H4Biopterin (pM)

Figure 5 . Effects of H4B on the inhibition by catalase (800 unitsiml) of N O synthesis (open symbols) and leishmanicidal activity (closed symbols) of macrophages activated with IFN-y (400 unit/ml) and LPS (10 ng/ml). Control cultures were activated macrophages incubated with (triangles) or without (squares) catalase in the abscnce of H4B.

4 Discussion

Data reported here demonstrate that the production of NO and the elimination of intracellular leishmania by activated macrophages can be inhibited by catalase. Since catalase converts H202 to molecular oxygen, these results suggest that H202 may be involved in both NO synthesis and killing of the intracellular parasites by maci-ophages. However, earlier reports show that inhibition of leishmanicidal activity and NO synthesis by L-NMMA was accompanied by enhancement of the production of H202 [24, 301, suggesting strongly that HzOz is not directly involved in the killing of this intracellular parasite. The finding here that

GPX, which effectively removed H202 in this system, yet did not influence the NO synthesis or the leishmanicidal capacity of the macrophages, is consistent with this inter- pretation.

It is at present unclear how exogenously added catalase affects the intracellular NO synthesis. This enzyme could exert its influence by modulating the oxygen tension in the culture medium or, alternatively, it could have been pinocytosed into the macrophages and thus affects the NO synthesis system more directly within the cytoplasm. This latter possibility may account for the relatively high concentrations of the enzyme required to demonstrate its effect in vitro. It may also explain the apparent difference between the macrophage system and the endothelium and neutrophil systems where catalase was reported not to affect NO synthesis [31], since these cells would not have the same pinocytic capacity as macrophages.

Earlier studies [32, 331 have demonstrated that SOD,which converts superoxide to H202, can effectively enhance vascular relaxation and the half-life of NO. This was interpreted as the result of the removal of 0, by SOD and thus protects NO from breakdown as a result of interaction with 0,. We have also found that, in the present system, SOD also increased the NO, levels in the culture supernatants and the leishmanicidal activity of activated macrophages, in a dose- and time-dependent manner (data not shown). Furthermore, the addition of SOD (800 U/ml) in the culture prevented the inhibitory effect of catalase (800 U/ml) (data not shown). SOD also had no effect on the induction of NO synthase in the activated macrophages (data not shown). These results, therefore, support the interpretation above [32, 331 that SOD protects and enhances the half-life of NO. The mechanism of the inhibition of NO synthesis by catalase, however, may be different.

The inhibition of NO synthesis and leishmanicidal activity of macrophages by catalase is unlikely to be due to the nonspecific action of contaminants in the catalase prepara- tions. Several batches of catalase with different degrees or purity gave identical effects on the basis of unit enzyme activity. Heat-inactivated enzyme was devoid of inhibitory activity, indicating that the effect is unlikely to be mediated by the possible contamination of transitional metals. Furthermore, catalase had no effect on the induction of NO synthase in activated macrophages. In contrast, the inhibi- tory effect of catalase was completely reversed by the presence of H4B in a dose-dependent manner. These data suggest that the effect of catalase on the macrophage activities described here may be due to the oxidation of this co-factor as a result of the enzymatic activity of catalase. The requirement for H4B as a cofactor in the generation of NO by murine macrophages is now well documented [9, 101, and H4B can readily penetrate the cellular mem- brane [34].

The synthesis of NO involves a number of cofactors besides H4B, including NADPH and FAD. Whether the redox reaction involving catalase also affect these other cofactors remains to be investigated. However, the fact that the effect of catalase can be fully reversed by H4B suggests that the oxidation of H4B plays an important role in the inhibition of NO synthesis in the present system. Whatever the precise

446

mechanism, these data demonstrate the interaction between reactive oxygen intermediates and the pathway leading to NO synthesis in activated macrophages.This may have important implications for the understanding and control of the antimicrobial potential of this pervasive nonspecific defense mechanism.

Y. Li, A. Severn, M.V. Rogers et al. Eur. J. Immunol. 1992. 22: 441-446

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