European Journal of Clinical Investigation (1991) 21, 3 16-322

ADONIS

00 1429729 100045V

Glutathione metabolism in activated human neutrophils: stimulation of glutathione synthesis and consumption of glutathione by reactive oxygen species M. BILZER & B. H. LAUTERBURG, Department of Clinical Pharmacology, University of Berne, Berne, Switzerland Received 29 June 1990 Abstract. Since glutathione (GSH) is involved in the modulation of the function of polymorphonuclear leucocytes (PMN) such as phagocytosis and production of reactive oxygen species, the metabolism of GSH was studied in human PMN. The concentration of GSH in resting PMN amounted to 13.3 nmol lo-’ PMN and remained stable over 100 min of incubation. Upon activation of PMN with phorbol myristate acetate intracellular GSH decreased to 50% of the resting concentration within 80 min. In the presence of buthionine sulfoximine, which inhibits the synthesis of GSH, the depletion of intracellular GSH was dramatically accelerated, indicating that activation of PMN is associated with a marked stimulation of GSH synthesis. Since a similar depletion of GSH was seen in the presence of propargylglycine, an inhibitor of the cystathionine pathway, most of the cysteine required for the resynthesis of GSH must originate from methionine and not from cysteine generated by the catabolism of GSH. Further studies showed that GSH is sequentially oxidized by 0;. and HOCI, first to GSSG and then to an unidentified compound, most likely a chloramine. In the presence of an adequate supply of GSH and NADPH which is required for the reduction of GSSG by glutathione reductase this further oxidation of GSSG was prevented. Thus, the highly toxic HOCl generated by PMN can be detoxified by the glutathione reductase system. The capacity of PMN to re-synthesize GSH may be an important determinant of PMN function.

Keywords. Glutathione metabolism, glutathione synthesis, hypochlorous acid, polymorphonuclear leucocytes. Introduction

Activated neutrophils release proteinases and oxygenderived species such as OF‘, H202 and hypochlorous acid (HOCI) which is generated via the myeloperoxidase-catalysed reaction of H202with chloride ions [lCorrespondence: Dr B. H. Lauterburg, Department of Clinical Pharmacology, Murtenstrasse 35, CH-3010 Berne, Switzerland.

316

31. Following activation, polymorphonuclear 1,wcocytes (PMN) and their environment are thus subjected to a massive oxidant stress. The reactive metabolites of molecular oxygen play a useful role in bacterial killing [4]. They may, however, oxididatively modify proteins in neutrophils and surrounding tissues [1,5-71 and thereby impair the function of PMN [8] and result in local tissue destruction [ 1,7,9- 121. Glutathione which is present intra- and extracellularly plays an important role in the detoxification of reactive oxygen species. It is a substrate in the glutathione peroxidase-catalyzed detoxification of H202, and reacts directly with 0;’ [13-151. Glutathione moreover may be involved in the detoxification of HOC1. The release of 01’and H202 is impaired in glutathione-depleted neutrophils [16]. In neutrophils of patients with cirrhosis there is a significant negative correlation between glutathione content and H202 production [17]. In addition, the production of peptido leukotrienes is decreased in glutathione-depleted macrophages [18]. Moreover, glutathione reductase, and thus the availability of reduced glutathione, has been shown to play an important role in the functional integrity of PMN [19-2 I]. Decreased concentrations of glutathione have also been associated with defects in microtubule assembly and microtubule-dependent processes such as phagocytosis [22,23]. Since the concentration of glutathione in PMN plays an important role in the modulation of the function of neutrophils, a better understanding of glutathione metabolism in activated PMN is of potential clinical relevance. The following experiments were performed to gain more insight into the effects of activation on glutathione metabolism in PMN and on the reactions of secretory products of PMN with glutathione. Our data show that activation of PMN is followed by depletion of intracellular glutathione and by a marked stimulation of glutathione synthesis in neutrophils and that glutathione (GSH) is sequentially oxidized to glutathione disulphide (GSSG) and then to an unknown product, most likely a chloramine or further-oxidized forms of glutathione.

GLUTATHIONE METABOLISM IN NEUTROPHILS Subjects and methods Isolation of human polymorphonuclear leucocytes ( P M N ) Humans PMN were isolated from venous blood obtained from healthy volunteers by sedimentation in a dextran solution and subsequent centrifugation on a Ficoll-Hypaque gradient for 20 rnin at 250 x g 1241. The purity of the isolated cells which was assessed by light microscopy routinely exceeded 96% and the viability assessed by Trypan blue exclusion was greater than 97%. The volunteers consented to donate their cells for the study which had been approved by the local ethics committee.

Incubations Cells were incubated in Hanks' balanced salt solution (HBSS)at aconcentrationof3 x 106cellsml-' at 37°C. Following a preincubation period of 30 min, PMN were stimulated by exposure to phorbol myristate . time of acetate (PMA, final concentration 1.2p ~ ) The stimulation corresponds to time zero of the incubation time. In some experiments the synthesis of glutathione was inhibited by adding buthionine sulfoximine (BSO), an inhibitor of y-glutamylcysteine synthetase 1251, at a concentration of 0.2 mM 30 min prior to stimulation of the PMN, To investigate the role of methionine for the synthesisof glutathione in activated PMN, cells were incubated in the presence of propargylglycine (PG), an inhibitor of the cystathionine pathway [26], at a concentration of 0.3 mM. To study the reaction of PMN-derived metabolites of oxygen with glutathione, PMN were incubated in 100 p~ GSH and 50 p~ GSSG, respectively. In some incubations NADPH (final concentration 1.O mM) with and without glutathione reductase (final activity 10 U ml-') were added in order to elucidate the sequence of reactions of PMN-derived oxidants with GSH. Catalase (final concentration 1000 U ml-I) which prevents the accumulation of H202, one of the substrates of myeloperoxidase, or dapsone (final con, inhibitor of myeloperoxidase 181, centration 30 p ~ ) an were added to additional incubations in order to define the role of PMN-derived HOCI in the oxidation of GSH. Finally, a cell-free system was used to investigate the reaction of GSH with HOCI. Sodium hypochlorite was added at various concentrations to GSH and GSSG, respectively, in HBSS buffer at 37°C. After 10 rnin the incubations were chilled on ice and assayed for total glutathione, GSH and GSSG. Assays For the determination of total intracellular glutathione, 0.5 ml of ice-cold HBSS were added to 0.5 ml of the incubations. Following centrifugation the supernatant was collected and acidified with 200 p1 2 M perchloric acid for the determination of extracellular glutathione. For the assay of intracellular glutathione

3 17

the cell pellet was lysed with 200 pl of Na-deoxycholate (0.6 rng ml- ') and acidified with 50 p1 of 2 M perchloric acid. Following another centrifugation step the supernatant was neutralized with KOH/K2C03 (both 0.3 mM), and total glutathione, i.e. GSH and GSSG, was measured by the kinetic assay of Tietze [27] in a double beam spectrophotometer at 412 nm. To 750 pl phosphate buffer (0.1 M, pH 7.4) containing 5 mM EDTA were added 100 pl of the neutralized supernatant, 50 pl of a 5 mM solution of NADPH, and 50 pl of a 1 mM solution of 5,5'-dithiobis-(2-nitrobenzoic acid). After equilibration at 25°C the test was started by adding 0-6 U glutathione reductase. A duplicate of each sample was measured with an internal standard of GSSG to control for potential inhibitors of the enzymatic reaction. Control experiments where 2-7 nmol GSH were added to cell pellets showed a recovery of 1005 1 5% (mean k SD, n = 6). Extracellular reduced glutathione was assayed by following the formation of the conjugate between GSH and l-chloro-2,4-dinitrobenzeneat 340-400 nm after addition of GSH S-transferase [28]. GSSG was measured by its reaction with NADPH catalysed by glutathione reductase [29]. GSSG concentrations are expressed in GSH-equivalents. The sum of extracellular GSH and GSSG determined by these assays was in good agreement with the results of the determination of total glutathione by the recycling assay of Tietze. Sodium hypochlorite. Commercial Na-hypochlorite was assayed by following the oxidation of 5-thio-2nitrobenzoic acid (TNB) to 5,5'-dithiobis-(Znitrobenzoic acid) (DTNB) at 412 nm [8]. TNB was synthesized by reduction of DTNB with excess sodium borohydride [8]. Superoxide release by PMN. OF' production of stimulated PMN at 37°C was measured by following the OF-dependent reduction of cytochrome c at 550 nm [24]. All chemicals were obtained from Sigma Chemical Co, St. Louis, MO 63178, USA. Statistical analysis. All results are expressed as mean +_ standard deviation. Group means were compared using Student's t-test for unpaired samples. Results

Intracelluhr GSH The intracellular concentration of total glutathione was 13.3f2.3 nmol lo PMN (n = 11). Assuming an intracellular water space of 3.47 pl PMN [30] and equal distribution of glutathione throughout this space, the intracellular concentration thus amounts to approximately 4 mM. Under resting conditions the GSH concentration in PMN remained stable for 100 min (Fig. 1). In contrast, the GSH concentration remained stable for only 20 min upon stimulation with PMA whereupon it decreased progressively, reaching approximately 50% of the initial concentration after

~'

318

M. BILZER & B. H. LAUTERBURG

I

I

15

4

.-

0

t

10

2

t

c

l

k

5

u 0

0 0

Ec

40

80

2 4

Incubation t i m e (min) Figure 1. lntracellular total glutathione (GSH plus GSSG) in human polymorphonuclear leucocytes during incubation in HBSS buffer for 100 rnin. The glutathione content remained stable in cells incubated in buffer alone (0). Stimulation of PMN by phorbol myristate acetate (PMA) was followed by the production of 0;.(A), and after a lag time of approximately 20 rnin by a progressive decrease in intracellular glutathione ( 0 ) .The concentration of total glutathione was significantly ( P < 0.01) lower in stimulated PMN from 40 rnin onward (mean fSD of six incubations).

0

40

80

Incubation time (min)

Figure 2. PMN incubated with an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO, O ) , and propargylglycine (PG, M), an inhibitor of the cystathionine pathway, respectively. In the presence of BSO the intracellular concentration of glutathione remained stable if the cells were not stimulated (0).In contrast to PMN with intact glutathione synthesis (Fig. 1) the concentration of GSH started to decline immediately upon stimulation with phorbol myristate acetate (PMA) at time zero, although the production of 02.’(A) was identical to the production of cells not exposed to buthionine sulfoximine (Fig. 1). lntracellular glutathione was significantly (P < 0.02) more depleted from 20 min onward in cells pretreated with BSO or PG than in the absence of BSO or PG as shown in Fig. 1. MeankSD of six incubations in each group.

80 min. Since more than 60% of the OF’ generated by the PMN was produced within the first 20 rnin the stabie concentration of GSH during that period suggests that the consumption of GSH evident during the later part of the experiments was compensated by re-synthesis during the initial phase of the oxidative burst. In the presence of BSO which inhibits the synthesis of GSH, there was indeed a dramatic acceleration of

the intracellular depletion of GSH (Fig. 2). After 100 rnin the PMN exposed to BSO and PMA contained less than 5% of the initial concentration of GSH, indicating that in the absence of BSO substantial quantities of GSH are newly synthesized in activated but not in resting PMN. The time-course of the decrease in intracellular glutathione paralleled that of OF‘ production which was similar in the presence (0.50 0.15 nmol/min X 1O5 PMN, n = 3) and absence (0.49k0.21 nmol/min x lo’ PMN) of BSO. In resting PMN the concentration of total glutathione remained stable also in the presence of BSO, indicating that GSH turnover is low in non-stimulated PMN. To rule out the possibility that prevention of the depletion of GSH by BSO was due to the scavenging of reactive oxygen species by the amino acid analogue BSO, PMN were incubated in the presence of 16 mM glycine. Depletion of intracellular glutathione by PMA stimulation was not affected by extracellular glycine, indicating that the effect of BSO cannot be attributed to a non-specific reaction of reactive oxygen species with amino acids. Incubation of PMN prior to stimulation with propargylglycine which inhibits the cystathionine pathway and thus prevents the cell from utilizing methionine for the synthesis of cysteine and subsequently GSH, also resulted in a significantly larger depletion of GSH than incubations with PMA alone (Fig. 2), indicating that a large fraction of the cysteine used for the synthesis of new GSH originates from methionine and not from the breakdown of GSH into its amino acid constituents. SOD and catalase in the incubation medium did not prevent the loss of intracellular glutathione upon stimulation (Table I), indicating that the accumulation of oxidants in the medium and phagocytic vacuoles during the oxidative burst is not responsible for the depletion of glutathione in PMN. Supporting this conclusion, the addition of two oxidants, Hz02 and diamide, to resting cells did not result in marked decreases in intracellular glutathione. Most likely, oxidants are released into the cytoplasm of PMN where catalase and SOD do not gain access.

Reaction of oxygen metabolites of P M N with glutathione

As shown in Table 1 the extracellular concentration of glutathione amounted to 5% of the intracellular concentration and remained stable during the incubation of non-stimulated PMN consistent with the observation that glutathione turnover is low in resting cells (Fig. 1 and 2). The stimulation of PMN by PMA which leads to depletion of intracellular glutathione did not increase the extracellular concentration of glutathione. No extracellular GSH or GSSG was detectable when the PMN were stimulated. Addition of superoxide dismutase and catalase did not result in measurable concentrations of extracellular GSH or GSSG. Exogenous GSH added at a concentration of 0.01 mM to PMN was consumed by stimulated, but not

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GLUTATHIONE METABOLISM IN NEUTROPHILS Table I. Reduced plus oxidized glutathione Intracellular (nmol lo-’ PMN) Conditions

0 min

100 min

Resting PMN + PMA + PMA + BSO 0.2 mM +PMA+SOD(66Uml-1)/catalase(130U ml-I) + diamide 0.2 mM + H202 0.25 mM + H202 0.50 mM +GSH 0.01 mM +PMA+GSH 0.01 mM

13.7f3.0 12.9+2.4 12.4+ 2.9 14.2+0.9 13.3+1.6 13.6+ 1.6 13.6+ 1.6 13.7+3,3 13.4+3.7

Extracellular ( p ~ )

___

n

-

0 min

100 min

n -

134+2.8 6.0+2,0 0.6 + 0.6 6.7+3,4 11.6+2.0 10.4+ 1.3 10.6+ 1.8 14.1+1,9 3.9+ 1.3

0.40+ 0.08 0.24 0.08 0.27 0.08 0.28 0.34+0.04 0.38 0.03 0.38 0.03 12-2+0.8 11.3+2,4

0.32+0.15 ND ND ND 0.39 + 0.05 0.25 f 0 . 2 I 0.34+0,18 11.4+ 0.40 0.4 +0.40

+ +

+ +

6 4 3 2 4

4 3 4 4

n.d. = not detectable.

c

1

3100

a 0 cn W

+

I

a -

50

0

W

B

P +

G

o

-

1

-I r

7

0

30

60

~~

+P

0

Figure 3. Incubation of PMN in 0.1 mM GSH. Total glutathione (0) remains stable over 90 min in the presence of resting PMN. The small decrease in GSH (A)is accounted for by a corresponding increase in GSSG (expressed as GSH equivalents, 0).When PMN were activated with PMA, GSH (A) rapidly disappeared. Its disappearance was only in part accounted for by an increase in GSSG )(,. such that total glutathione (m) disappeared, indicating that a new species of glutathione is formed. Mean+ SD of four incubations.

T

-

60

7

90

Incubation time (min)

90

Incubation time (min)

30

Figure 5. Incubation of PMN in 0.05 mM GSSG (corresponding to 0.1 mM GSH equivalents). With resting cells, the concentration of GSSG remains stable (0) whereas extracellular GSSG is rapidly consumed ( 0 ) when cells are stimulated with PMA MeanFSD, n=3.

--

50

0 3 01

e

+ x

w

o

Y0 O

30

60

90

Incubation t i m e (min) $

oJ , 0

30

Incubaiion

60 time bin)

90

Figure 4. Activation by PMA ofPMNincubated in 1 0 0 GSH ~ ~ plus NADPH (1 mM) and glutathione reductase (10 U ml-I). In comparison to the data shown in Fig. 3 little GSSG ( 0 )is detectable because of the presence of glutathione reductase and NADPH. The decrcase in total glutathione (m) and GSH (A) is virtually abolished, indicating that the new species of glutathione is formed from GSSG. Mean+SD, n = 3 .

Figure 6. Consumption of extracellular glutathione by PMN preincubated with catalase (1000 U ml-I), which prevents the accumulation of HzOz, one of the substrates of myeloperoxidase (U), or dapsone (30 p ~ which ) inhibits myeloperoxidase (0)following stimulation with PMA. Compared to cells that were not pretreated (Fig. 3) the consumption of total glutathione was significantly ( P i 0.01) decreased. Substantial quantities of GSSG were formed by cells pretreated with catalase ( 0 )and dapsone (0).Since neither catalase nor dapsone interfere with the generation of 0 2 the generation of GSSG under these conditions is best explained by the reaction of 0 2 ’ with GSH. MeanfSD, n=4.

320

M. BILZER & B. H. LAUTERBURG I -

0

HOCL (PLY)

Figure 7. Reaction of GSH (A) and GSSG (expressed as GSH equivalents, 0)with HOCI. With concentrations of HOCl ranging from 10 to 40 ~ L the M concentrationof GSSG was significantly lower (P< 0.01) than the concentrationof GSH. Compared to GSSG two times as much HOCl is required to consume equimolar amounts of GSH. HOCl reacts first with GSH to form GSSG and subsequently reacts with GSSG to form an unidentified compound, most likely a chloramine. Mean&SD, n=4.

by resting cells (Table I), suggesting that extracellular glutathione is degraded by PMN-derived products generated during the oxidative burst. In the presence of much higher concentrations of extracellular GSH (0.1 mM), the concentration of GSSG increased rapidly upon stimulation of the PMN (Fig. 3). However, the disappearance of GSH was only partially accounted for by the increase in GSSG, suggesting that an additional pathway consuming GSH and/or GSSG must be involved. In the presence of extracellular glutathione reductase (10 U ml-I) and NADPH (1.0 mM), total GSH remained virtually stable in spite of the activation of the PMN (Fig. 4). This could not be attributed to scavenging of reactive oxygen species by NADPH since the disappearance of extracellular GSH was identical to control incubations when cells were incubated with NADPH without glutathione reductase. Thus, the substrate for the reaction responsible for the disappearance of total glutathione appears to be GSSG rather than GSH. One million PMN incubated in 0.05 mM GSSG consumed 23.5 & 1a 8 (mean & SD, n = 3) nmol GSHequivalents in 60 min, i.e. during the respiratory burst (Fig. 5). Catalase (1000 U ml-I) which destroys H202, the substrate for myeloperoxidase, and dapsone (30 p ~ ) ,which inhibits myeloperoxidase, markedly decreased the consumption of extracellular glutathione by activated PMN, indicating that myeloperoxidasederived oxidants were in part responsible for the loss of glutathione (Fig. 6). However, the generation of GSSG was only partially prevented by catalase and dapsone (Fig. 6). Since neither catalase nor dapsone interfere with the generation of 0;' the formation of GSSG under these conditions is best explained by the reaction of 0 2 ' with GSH. These observations suggested that myeloperoxidase-derived oxidants were mainly responsible for the disappearance of GSSG. Incubation of GSSG with

0.5 HOCl (mu)

1.o

Figure 8. Reaction of HOCl with GSH. Incubation of excess GSH with HOCl results in the disappearance of GSH (A) and the stochiometric appearance of GSSG (a).

various concentrations of HOCl indeed resulted in a concentration-dependent disappearance of GSSG (Fig. 7). Similar results were obtained with GSH, but higher concentrations of HOCl were necessary to consume all the glutathione, suggesting that GSH is first oxidized to GSSG by HOCl and the GSSG thus formed is then further oxidized to an unknown compound, most likely a chloramine (Fig. 8). Discussion The present data show that activation of human PMN by PMA results in depletion of intracellular GSH which is initially compensated for by a marked increase in the synthesis of the tripeptide. In normal PMN it took 80 min following stimulation to decrease the intracellular concentration of GSH by 50%, whereas in cell lacking the capability to synthesize new GSH the half-life of GSH was 22 min. A similar stimulation of GSH synthesis upon activation has also been observed in peritoneal macrophages from rats [3 I]. Under the present experimental conditions the increased rate of synthesis was capable of maintaining the intraceliular concentration of GSH for approximately 20 min, although the generation of oxidants by the stimulated cell is maximal during this period of time. Later, substrate availability may become rate limiting and the concentration of GSH progressively decreases due to its reaction with oxidants. The magnitude of the respiratory burst of PMN in vivo is not known, and in uitro it varies with the stimulus used. With physiological stimuli the compensatory increase in GSH synthesis may be an important mechanism to maintain adequate intracellular concentrations of GSH. An increased availability of GSH could contribute to the protection of PMN from oxidative injury throughout the respiratory burst, thus permitting the PMN to function and sustain an oxidant milieu in phagocytic vacuoles long enough to degrade phagocytosed material. Genetic deficiency of glutathione synthetase indeed results in oxidative damage to neutrophils [32]. The experiments with propargylglycine which pre-

GLUTATHIONE METABOLISM IN NEUTROPHILS vents the cell from utilizing methionine for the synthesis of GSH via the cystathionine pathway [26], indicate that much of the cysteine required for the stimulation of GSH synthesis in activated PMN originates from methionine. Thus, the formation of cysteine from methionine via the cystathionine pathway is important not only in hepatocytes [33] but other cells as well. Since depletion of intracellular GSH impairs neutrophi1 functions such as phagocytosis and bactericidal activity [ 16,17,20], impaired transsulfuration and an impaired ability to re-synthesize the GSH in certain disease states such as cirrhosis [34,35] may be an important determinant of PMN function and could at least in part account for the impaired antimicrobial defence in some patients with liver disease [36]. The fate of GSH following activation of PMN is difficult to assess since intracellular GSH disappeared and neither GSH nor GSSG were recovered in the medium of activated PMN. Considering the physicochemical properties of the oxidants produced by PMN, however, the reactions of intra- and extracellular glutathione may be expected to be identical. Therefore, the fate of GSH was studied in more detail by adding glutathione to the incubation medium. The experiments with high extracellular concentrations of GSH and GSSG, respectively, demonstrate that large quantities of glutathione are consumed by activated PMN. In the process GSH appears to be sequentially oxidized, first to GSSG and subsequently to an unidentified product, most likely a chloramine and possibly further-oxidized forms of glutathione. Two processes appear to be responsible for this oxidation, the reaction with 0;' and the generation of myeloperoxidase-derived oxidants. In the presence of catalase and dapsone which both prevent the formation of products of myeloperoxidase [8] there was still a substantial formation of GSSG (Fig. 6) indicating that the oxidation of GSH occurs at least in part by OF' released by activated PMN. Myeloperoxidase-derived oxidants are involved as well since catalase which removes extracellular HzOz and thereby limits the availability of the substrate for myeloperoxidase, and dapsone, an inhibitor of myeloperoxidase, largely prevented the oxidation of glutathione by activated neutrophils. Since extracellular (and thus also intravesicular) scavengers of 0;' and H202 like SOD and catalase did not prevent the depletion of GSH (Table I), these oxidants must be secreted not only into the extracellular space but also into the cytoplasm where the scavengers do not gain access. The respiratory burst is associated with a marked increase in NADPH consumption. In addition to being utilized for the reduction of molecular oxygen which is required for the generation of OC', NADPH participates in the reduction of GSSG to GSH via glutathione reductase. The present data indicate that in the presence of glutathione reductase and NADPH the irreversible loss of GSH associated with the activation of PMN can be largely prevented. Under these conditions GSSG is rapidly reduced to GSH and no GSSG is

32 I

available for further oxidation by neutrophil-derived oxidants. Thus, two processes limit the irreversible loss of GSH from PMN during the respiratory burst, stimulation of synthesis and reduction of GSSG. The detoxification of myeloperoxidase-derived oxidants by GSH prevents the formation of toxic chloramines [37,38]. In contrast to other scavengers of HOCl such as taurine, the oxidation of GSH by HOCl does not generate a 'long-lived oxidant' as long as an adequate supply of GSH is maintained by re-synthesis and GSSG can be reduced via glutathione reductase.

Acknowledgments Supported by grant number 3.812-0.87 from the Swiss National Foundation for Scientific Research and Deutsche Forschungsgemeinschaft. The technical assistance of Ms E. Junker and Ms Th. Schaefer is gratefully acknowledged. References 1 Henson PM, Johnston RB. Tissue injury in inflammation. Oxidants, proteinases, and cationic proteins. J Clin Invest 1987;79:669- 74. 2 Zgliczynski JM, Stelmaszynska T, Domanski J, Ostrowski W . Chloramines as intermediates of oxidation reaction of amino acids by myeloperoxidase. Biochim Biophys Acta 1971;235:41924. 3 Baggiolini M, Bretz U, Dewald B, Feigenson ME. The polymorphonuclear leukocyte. Agents Action 1978;8:3-10. 4 Stossel TP. Phagocytosis. N Engl J Med 1974;290:717-23,77480,833- 9. 5 Oliver CN. Inactivation of enzymes and oxidative modification of proteins by stimulated neutrophils. Arch Biochem Biophys I987;253:62-72. 6 Beck-Speier I, Leuschel L, Luippold G, Maier KL. Proteins released from stimulated neutrophils contain very high levels of oxidized methionine. FEBS Lett 1988;227:1-4. 7 Johnson RJ, Couser WG, Chi EY. Adler S, Klebanoff SJ. New mechanism for glomerular injury: myeloperoxidase-hydrogen peroxide-halide system. J Clin Invest 1987;79:1379-87. 8 Thomas EL, Grisham MB, Jefferson MM. Myeloperoxidase dependent effect of amines on functions of isolated neutrophils. J Clin Invest 1983;72:44-54. 9 Weiss SJ, Regiani S . Neutrophils degrade subendothelial matrices in the presence of alpha-I-proteinase inhibitor. Cooperative use of lysosomal proteinases and oxygen metabolites. J Clin Invest 1984;73:1297-303. 10 Cathcart MK, Morel DW, Chisolm I11 GM. Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic. J Leukocyte Biol 1985;38:341-50. I 1 Fox RB. Prevention of granulocyte-mediated oxidant lung injury in rats by a hydroxyl radical scavenger, dimethylthiourea. J Clin Invest 1984;74:1456-64. 12 Visser MCM, Winterbourn CC, Hunt JS. Degradation of glomerular basement membrane by human neutrophils in viw. Biochem Biophys Acta 1984;804:154-60. 13 Wefers H, Sies H. Oxidation of glutathione by the superoxide radical to the disulfide and the sulfonate yielding singlet oxygen. Eur J Biochem 1983;137:29-36. 14 Ross D, Cotgreave I, Moldeus P. The interaction of reduced glutathione with active oxygen species generated by xanthineoxidase catalyzed metabolism of xanthine. Biochim Biophys Acta 1985;841:278-82. 15 Thomas EL, Learn DB, Jefferson MM, Weatherred W. Superoxidedependent oxidation of extracellular reducing agents by isolated neutrophils. J Biol Chem 1988;263:2178-86. 16 Younes M, Robke A. Inhibition of granulocyte-mediated release

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of oxygen free radicals following glutathione depletion. Toxicol Lett I988;41: 139-43. 17 Rajkovic IA, Williams R. Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis. Hepatology 1986;6:252-62. 18 Rouzer CA, Scott WA, Griffith OW, Hamill AL, Cohn ZA. Depletion of glutathione selectively inhibits synthesis of leukotriene C by macrophages. Proc Natl Acad Sci USA 198 1;78:2532-6. 19 Roos D, Weening RS, Voetman et nl. Protection of phagocytic leukocytes by endogenous glutathione: studies in a family with glutathione reductase deficiency. Blood 1979;53:85 1-66. 20 Cohen HJ, Tape EH, Novak J, Chovaniec ME, Leigy P, Whitin JC. The role of glutathione reductase in maintaining human granulocyte function and sensitivity to exogenous H202. Blood 1987;69:493-500. 21 Baehner RL, Boxer LA, Allen JM. Autooxidation as a basis for altered function by polymorphonuclear leukocytes. Blood 1977:50327-35. 22 Burchill BR, Oliver JM, Pearson CB, Leinbach ED, Berlin RD. Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes. J Cell Biol 1978;76:439-47. 23 Oliver JM, Albertini DF, Berlin RD. Effects of glutathioneoxidizing agents on microtubule assembly and microtubuledependent surface properties of human neutrophils. J Cell Biol 1976;71:921-32. 24 Markert M, Andrews PC, Babior BM. Measurement of 0 2 production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils. Meth Ezymol 1984;105:358-65. 25 Griffith OW. Mechanisms of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione biosynthesis. J Biol Chem 1982;257:1370412. 26 Abeles RH, Walsh CT. Acetylenicenzyme inactivators. Inactivation of cystathionase, in uitro and in rriuo, by propargylglycine. J Am Chem Soc 1973;95:6124-5.

27 Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal Biochem 1969;27:502-22. 28 Brigelius R, Muckel C, Akerboom TPM, Sies H. Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide. Biochem Pharmacol 1983;32:2529-34. 29 Akerboom TPM, Sies H. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Meth Enzymol 1981;77:373-82. 30 Hawkins RA, Berlin RD. Purine transport in polymorphonuclear leukocytes. Biochim Biophys Acta 1969;l73:324 37. 31 Rouzer CA, Scott WA. Griffith OW, Hamill AL, Cohn ZA. Glutathione metabolism in resting and phagocytizing peritoneal macrophages. J Biol Chem 1982;257:2002-8. 32 Spielberg SP, Boxer LA, Oliver JM, Allen JM, Schulman JD. Oxidative damage to neutrophils in glutathione synthetase deficiency. Br J Haematol 1979;42:215-23. 33 Reed D, Orrenius S. The role of methionine in glutathione biosynthesis by isolated hepatocytes. Biochem Biophys Res Commun 1977;77:1258-64. 34 Horowitz JH, Rypins EB, Henderson JM er al. Evidence for impairment of transsulfuration pathway in cirrhosis. Gastroenterology 1981;81:668-75. 35 Burgunder JM, Lauterburg BH. Decreased production of glutathione in patients with cirrhosis. Eur J Clin Invest 1987;17:40814. 36 Rajkovic IA, Williams R. Mechanisms of abnormalities in host defences against bacterial infection in liver disease. Clin Sci 1985;68:247-53. 37 Weiss ST, KIein R, Slivka A, Wei M. Chlorination of taurine by human neutrophils. Evidence for hypochlorous acid generation. J Clin Invest 1982;70:598-607. 38 Grisham MB, Jefferson MM, Melton DF, Thomas EL. Chlorination of endogenous amines by isolated neutrophils. Ammoniadependent bactericidal, cytotoxic, and cytolytic activities of the chloramines. J Biol Chem 1984;259:10404- 13.