620 Biochimica et Biophysica Acta, 585 (1979) 620--629 © Elsevier/North-Holland Biomedical Press
BBA 28971
THE EFFECT OF OXIDANT STRESS ON DIAMIDE-TREATED HUMAN GRANULOCYTES
S T E P H E N J. WEISS * and A R T H U R
L. S A G O N E , Jr.
Division of Hematology and Oncology, Ohio State UniversityCollege of Medicine, Columbus, O H 43210 (U.S.A.) (Received November 16th, 1978)
Key words: Neutrophil; Diamide; Oxidant stress; Chemiluminescence; Phagocytosis; Superoxide dismutase
Summary The role of sulfhydryls in the protection of human polymorphonuclear neutrophfls against extraceUular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant~enerating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xanthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants. Introduction
Granulocytes generate a host of potentially toxic oxygen intermediates during phagocytosis [1]. These compounds have been implicated in the bac* T o w h o m c o ~ e s p o n d e n c e s h o u l d be addressed at: S i m p s o n M e m o r i a l R e s e a r c h I n s t i t u t e , 1 0 2 O b s e r v a t o r y Lane, A n n A r b o r , MI 4 1 8 0 9 , U . S . A .
621
tericidal mechanism, inflammation and tumoricidal capability [2--5]. Harmful compounds generated include superoxide anion (O~-), hydrogen peroxide (H202) and the products of their interaction, the hydroxyl radical (OH') and singlet oxygen (O~) [1]. During the act of ingestion, these reactive substances are released into the phagocytic vacuole as well as the extracellular milieu. In order to protect itself from oxidant injury, the granulocyte is armed with a variety of detoxifying mechanisms. Superoxide is reduced to H202 by the enxyme superoxide dismutase [6], while H202 may be degraded by catalase, myeloperoxidase or the glutathione~lutathione peroxidase system [6,7]. However, as granulocytes phagocytize, there is a p r o m p t and nearly complete depletion of intracellular glutathione [8]. Thus, the post-phagocytic granulocyte may be rendered more susceptible to oxidant injury. In this study, we evaluated the capacity of granulocytes treated with the thiol-oxidizing agent, diamide [ 9,10 ], to withstand extracellular oxidant exposure. Materials and Methods
Xanthine, xanthine oxidase ( 1 2 5 U / m l ) , bovine superoxide dismutase (3000 U/rag) catalase (type C40), diamide, mannitol, benzoate, L-tryptophan, zymosan, and L-histidine were obtained from the Sigma Chemical Co., St. Louis, MO. The catalase was freed of contaminating superoxide dismutase by repeated washings over an XM-100 Diaflo ultrafiltmtion membrane from the Amicon Corp., Scientific Systems Div., Lexington, MA [11]. Selegman's balanced salt solution, Earle's balanced salt solution and Hank's balanced salt solution were purchased from Grand Island Biological Co., Grand Island, NY. Cell preparation. Venous blood was collected in EDTA from normal healthy volunteers. Granulocytes were isolated by dextran sedimentation. The granulocytes were further purified by Ficoll-Hypaque gradient centrifugation to remove contaminating mononuclear cells [12]. The final preparations conrained greater than 98% granulocytes and less than 2% mononuclear cells. Exposure to 02 metabolites. In order to expose the cells to extracellular oxygen metabolites, 20 • 106 polymorphonuclear neutrophils were suspended in Selegrnan's balanced salt solution with 10 -4 M xanthine and 7.5 • 10 -3 U/ml of xanthine oxidase for 40 rain (unless otherwise indicated) in the presence or absence of 25 nmol diamide/106 cells at 37 ° C. At the end of the incubation period, the cells were diluted in ice-cold buffer, washed (400 X g, 10 min), resuspended in Hank's balanced salt solution, recounted and immediately assayed for metabolic parameters. In some experiments, the following agents were added during the incubations: 1.0/~g/rnl superoxide dismutase (3000 units/mg), 250/~g/ml catalase, 10 mM mannitol, 20 mM benzoate, 40 mM ethanol, 10 mM tryptophan or 10 mM histidine. Glucose metabolism. Hexose monophosphate shunt activity was estimated from the production of labeled CO2 from [1-14C]glucose [13]. The generation of 14CO2 by granulocyte suspensions was measured continuously using the ionization chamberelectrometer m e t h o d as previously described [13]. 1--2. 107 granulocytes were suspended in 4 rnl of Earle's balanced salt solution with 50 rag/100 ml of glucose and 5/~Ci of [1-~4C]giucose and added to 25-ml triple-headed distilling flasks. The inlet of the metabolic flask was connected to
622
a gas cylinder containing compressed air with 5% CO:. The outlet arm of the flask was connected to a Cary-Tolbert ionization chamber and a Cary Model 401 vibrating reed electrometer (Cary Instruments, Fairfield, NJ). The third arm of the flask was covered with a rubber stopper through which reagents could be added. The incubation flask were stirred continuously. After baseline 14CO2 production was established, opsonized zymosan particles were prepared by the method of Webb et al. [14] and added in 0.4 ml of normal saline (4 • 10 s particles/ml). In some experiments, the cells were stimulated by the addition of 25--50 nmol diamide/10 ~ cells or the xanthine.xanthine oxidase system. Results were expressed as mV generated/107 cells. Formate oxidation. H202 generation was estimated by the generation of labeled CO2 from [14C]formate [13]. The method employed was identical to the glucose metabolism experiments except that 5/~Ci (50 Ci/mol) of [14C]formate was added in place of labeled glucose. Results were expressed as mV generated/107 cells. Phagocytosis. Phagocytosis was estimated morphologically by counting 100 cells following incubation with zymosan particles at 37°C. Aliquots of cells were removed and examined at 5, 10, 30 and 60 rain after the addition of zymosan particles. Results were expressed as per cent of control phagocytosis at 60 rain unless otherwise indicated. Chemiluminescence assay. Chemiluminescence was studied using a Packard Model 3225 liquid scintillation counter (Packard Instrument Co., Inc., Downers Grove, IL), in a dark-room as described by Webb et al. [14]. 1--2 • 107 granulocytes were suspended in 6.6 ml of Earle's balanced salt solution in siliconized liquid scintillation vials. Zymosan was added in 0.4 ml of saline as in the other experiments. Results
Exposure of polymorphonuclear neutrophils to the oxygen metabolites generated by the xanthine-xanthine oxidase system [ 11] was associated with a stimulation of both [1.14C]glucose oxidation and [14C]formate oxidation which was maximal by 30 rain and then gradually decreased toward baseline (Fig. 1). Thus, it appears that polymorphonuclear neutrophils can degrade the oxidants generated outside the cell by both glutathione peroxidase and catalase and suggests these enzymes may afford protection against oxidant stress. In order to determine if the exposure of the polymorphonuclear neutrophils to extracellular oxidants incurred any metabolic damage, the ability of these cells to oxidize [14C]formate, [1-14C]glucose, chemiluminesce and phagocytize was studied. Granulocytes exposed to the xanthine-xanthine oxidase system for 40 rain, washed, resuspended and incubated with a phagocytic stimulus (zymosan) performed comparably to untreated cells (Table I). Increasing the xanthine-xanthine oxidase concentration ten fold did not alter the results. As previously reported, exposure of neutrophils to diamide caused a marked stimulation of [1.14C]glucose oxidation [15] (Fig. 2). Presumably this stimulation is mediated by oxidation of intracellular glutathione followed by its reduction by glutathione reductase and NADPH generated by the hexose monophosphate shunt [7]. Exposure of polymorphonuclear neutrophils to diamide alone
623 3r
Xanthine
Oxidase = | A~ded "~ 2 t- X-nthine I J" I Added I /
|
-60
0
I0
20 30 40 TIME { rains.)
50
60
70
F i g . 1. E f f e c t o f x a n t h i n e - x a n t h i n e o x i d a s e s y s t e m o n t h e o x i d a t i o n o f [ 1-I 4 C ] g l u c o s e a n d [ 14 C ] f o r m a t e oxidation by polymorphonuclear neutrophil. The curves represent a continuous measurement of 14CO2 production from [l-14C]glucose (c 9) o r [ 1 4 C ] f o r m a t e (e ~) and were d~awn from the data p o i n t o f a single e x p e r i m e n t . A f t e r s t e a d y - s t a t e c o n d i t i o n s w e r e e s t a b l i s h e d , 1 0 -4 M x a n t h i n e w a s a d d e d f o l l o w e d b y t h e addition o f 9 . S • 1 0 - 3 U / m l o f x a n t h i n e o x i d a s e . T h e y - a x i s i n d i c a t e s t h e e l e c t r i c a l s i g n a l in m V g e n e r a t e d b y 1 4 C O 2 / 1 0 9 cells. T h e e x p e r i m e n t is t y p i c a l o f f o u r p e r f o r m e d .
did not stimulate [6-t4C]glucose oxidation, [14C]formate oxidation or chemiluminescence. Neutrophils incubated with diamide for 40 rain, washed and allowed to phagocytize performed comparably [1-14C]glucose, [14C]formate, chemiluminescence and phagocytosis) to untreated cells.Thus, it appeared that exposure of polymorphonuclear neutrophils to xanthine-xanthine oxidase or diamide alone did not impair the metabolic response associated with phagocytosis. In order to study the response of thiol~iepleted cells to withstand extracellular oxidants, granulocytes were simultaneously exposed to diamide and the xanthine-xanthine oxidase system for 40rain, washed, resuspended and allowed to phagocytize. The chemiluminescence response of these treated washed cells in response to opsonized zymosan was markedly impaired (Fig. 3). In eleven experiments, peak chemiluminescence was impaired 80 + 8%. The degree of impairment was time dependent, i.e. the greater the incubation period with the xanthine-xanthine oxidase system the greater the injury (Fig. 4). It was always necessary to incubate the cells simultaneously with the enzyme system and diamide in order to demonstrate the maximal impairment of the polymorphonuclear neutrophil. The ability of the cells to oxidize [1J4C]glucose or [14C]formate during phagocytosis was also significantly impaired (Figs. 5 and 6). In four experiments [14C]formate and [1J4C]glucose oxidation were inhibited 72 + 8% and 83 -+ 7%, respectively.
TABLE I METABOLIC STATUS OF XANTHINE-XANTHINE
OXIDASE-TREATED
GRANULOCYTES
A s s a y p e r f o r m e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . R e s u l t s e x p r e s s e d as m e a n ± S . D . o f f o u r e x p e r i ments. Assay
% of control
[1-14C]Glueose oxidation [14C]Formate oxidation Chemiluminescence Phagocytolis
96 100 102 98
± ~ + ±
5 4 3 4
624
2 ¸
i
diamide
II -60
I
,
0
l, 15
,
, I
I
i
I
I
I
30
45
60
75
90
105
time (minutes) Fig. 2. E f f e c t of d i a m i d e o n t h e o x i d a t i o n o f [ l - 1 4 C ] g l u c o s e b y p o l y m o r p h o n u c l e a r n e u t r o p h i l . The c u r v e s r e p r e s e n t a c o n t h l u o u s m e a s u r e m e n t o f 1 4 C O 2 p r o d u c t i o n f r o m [1 -I 4 C ] g l u e o s e a n d w e r e d r a w n f r o m t h e d a t a p o i n t o f a single e x p e r i m e n t . A f t e r s t e a d y - s t a t e c o n d i t i o n s were e s t a b l i s h e d , d i a m i d e (25 n m o l / 1 0 6 p o l y m o r p h o n u c l e a r n e u t z o p h i l ) was a d d e d t o o n e s u s p e n s i o n (o o ) as i n d i c a t e d b y t h e a r r o w , The s e c o n d s u s p e n s i o n (e -') w a s u s e d as t h e c o n t r o l . The y-axis i n d i c a t e s t h e e l e c t r l c a l signal i n m V g e n e r a t e d b y 1 4 C O 2 / 1 0 7 cells, The e x p e r i m e n t is t y p i c a l o f e i g h t p e r f o r m e d .
Although the metabolic response of the treated cells was significantly impaired, viability was judged greater than 95% by Trypan Blue exclusion. Examination of these cells after incubation with opsonized zymosan revealed
12
101
I0 w
~o 8 w
i6 4
21 I 7
I 14
I 21
I 28
I
I
35
7
I
/
I
l
14 21 28 35 time (minutes) time (minutes) Fig. 3. E f f e c t o f x a n t h i n e - x a n t h i n e o x i d a s e or x a n t h i n e - x a n t h i n e o x i d a s e plus d i a m i d e o n p o l y m o r p h o n u c l e a r n e u t ~ o p h i l c h e m i l u m i n e s c e n c e . P o l y m o r p h o n u c l e a r n e u t r o p h i l s w e r e i n c u b a t e d in b u f f e r a l o n e ( o - - - - - - . - ~ ) ; x a n t h i n e - x a n t h i n e o x i d a s e (a -') or x a n t h i n e - x a n t h i n e o x i d a s e and d i a m i d e ( i a) for 4 0 m i n , w a s h e d a n d t h e n r e s u s p e n d e d i n fresh buffer. The c h e m i l u m i n e s c e n c e g e n e r a t e d b y t h e p o l Y m o r p h o n u e l e a r n e u t r o p h i l s ( e p m / 1 0 6 ) d u r i n l t h e p h a g o c y t o s i s o f z y m o s e n p a r t i c l e s was t h e n d e t e r m i n e d . E a c h p o i n t is t h e m e a n o f t w o o b s e r v a t i o n s . The e x p e r l m e n t is t y p i c a l of 11 p e r f o r m e d . Fig. 4. E f f e c t o f t h e t i m e o f p r e i n c u b a t / o n w i t h the x a n t h i n e - x a n t h i n e o x i d a s e s y s t e m a n d d i a m i d e o n polymorphonuclear neutrophil chemiluminescence. Polymorphonuclear neutrophils were preincubated w i t h t h e x a n t h i n e - x a n t h i n e o x i d a s e s y s t e m a n d d i a m i d e for 1 5 r a i n ( ! -'), 3 0 r a i n (e -') or 4 5 rain (o ~), w a s h e d a n d t h e n r e s u s p e n d e d in fresh m e d i u m . The c h e m i l u m i n e s c e n c e o f t h e p o l y m o r p h o n u e l e a x n e u t r o p h i ] s ( c p m / 1 0 6 ) duldng the p h a g o c y t o s i s o f opsondzed z y m o s a n p a r t i c l e s w a s t h e n d e t e r m i n e d . The results r e p r e s e n t s a single e x p e r i m e n t w i t h is t y p i c a l of f o u r p e r f o r m e d . Ae i n d i c a t e d , ceils i n c u b a t e d w i t h x a n t h i n e - x a n t h i n e o x l d a s c a l o n e (o o) w e r e n o t i m p a i r e d (see Fig. 3).
625
12
~ 4
-
60
zymo~n added
0
10
20
30
40
50
60
time (minutes)
Fig. 5. Effect of x a n t h i n e - x a n t h i n e oxidase or x a n t h i n e - x a n t h i n e oxidase plus di a mi de on p o l y m o r p h o nuclear neutxophil hexose m o n o p h o s p h a t e shunt activity. P o l y m o r p h o n u c l e a r n e u t r o p h i l s were prelneub a t e d with x a n t h i n e - x a n t h i n e oxidase alone ( o - - - - - - - ~ ) or x a n t h i n e - x a n t h i n e oxidase plus di a mi de for 40 m i n ( e - --), washed and resusPended in fresh m e d i u m in a m e t a b o l i c flask w i t h [ l - 1 4 C ] g l u c o s e (see Materials and Methods). Curves represent a c o n t i n u o u s meamarement of 14CO2 p r o d u c t i o n from [ l - 1 4 c ] g l u c o s e . The y-axis indicates the electrical 'signal in m V generated by t he 14CO2. After steadystate c o n d i t i o n s were established, Opsonized z y m o s a n particles were a dde d as i ndi c a t e d b y t he arrows. The curves were d r a w n from the d a t a p o i n t of a single e x p e r i m e n t and are representative of four performed.
greater than 90% inhibition of phagocytosis. Metabolism studies with opsonized bacteria or latex particles were similar to experiments performed with zymosan. The ability o f the diamide-xanthine-xanthine oxidase-treated granulocytes to respond to a non-phagocytic stimulus of metabolism was studied by incubating the cell with a second dose of diamide and then measuring [1-14C]glucose oxidation (Fig. 7). In three experiments, [1-14C]glucose oxidation by the injured cells was 56 + 8% o f controls.
2.0
u
~ 1.0 O.5
0
10
20
30
40
50
60
time (minutes)
Fig. 6. Effect of x a n t h l n e - x a n t h i n e oxidase or x a n t h i n e - x a n t h i n e oxidase plus d i a m i d e on p o l y m o r p h o nuclear n e u t r o p h i l formate o x i d a t i o n . P o l y m o ~ h o n u e l e a r n e u t r o p h i l s were i n c u b a t e d w i t h xa nt hi ne x a n t h i n e oxidase alone (oo) or xaDthine-xanthine oxidase pl us d i a m i d e for 40 m l n (~~), washed and resuspended in fresh m e d i u m in a m e t a b o l i c flask w i t h [ 1 4 C ] f o n n a t e (see Materials and Methods). The eurves represent a c o n t i n u o u s m e a s u r e m e n t of 14CO2 p r o d u c t / o n from [ 1 4 C ] f o r m a t e , The y ~ indicates the electrical signal in m V generated b y t h e 14CO2. A ft e r stcady-state c o n d i t i o n s were establ/shed, opson/zed z y m o R n partleles were added as i n d i c a t e d b y t he arrows. The curves were d r a w n from the d a t a p o i n t s of a single e x p e r i m e n t and are representative of four pe rforme d.
626
i0I i"l:m./f "6()
0
10
20 30 tlme (minutes)
40
50
60
l~Ig. 7. Effect of o x i d a n t injury on the d/amide response of p o l y m o r p h o n u c l e a r neutrophfls, o . o, the o x i d a t i o n of [1-14C]illueose by p o l y m o r p h o n u c l e ~ neut~oph~lls p r e i n c u b a t e d w i t h xanthineoxanthine ox/dase, and • --, p r e t n ~ b a t e d w i t h xant~dne-xanthine oxidase and diamide. After steady-state condiflons were established, 50 n m o l / 1 0 6 ceils of d i a m k l e was added a s / n d i c a t e d b y t he arrows. The curves were d r a w n fro m th e d a t a p o / n t of a single e x p e r i m e n t and are representative of three performed.
In an attempt to determine the specific oxygen intermediate responsible for the damage, diamide-treated cells were incubated with various scavengers during their exposure to the xanthine-xanthine oxidase system. The cells were 16
14
12
0
7
14
21
28
35
time (minutes) F/g. 8. lhroteetive effect o f s u p m x i d e d i ~ ; u t a s e and c a t a l a n on the o x i d a n t inJm'y produced b y xantbtne- xan thine oxldmm ~ e s y s t e m . P o l y m o r p h o n u d e a r neutrophfls were I)reinoubated w i t h x a n t b l n e x a n t h l n e ox/dase (¢ --); x a n t h i n e - x a n t h i n e oxidase plu s d/am/de (s..-------.a); x a n t h i n e - x a n t h i n e oxidase, diamid e and s u p e r o x l d e d/mnutaas (o------.--¢); or x a n t h / n e - x a n t h / n e ox/dmm, d/m~ide and catalase (c~---------~). The cells were t h a n washed and the chemllumtnesolmce generated d u r l n l t he pha goc yt os / s of z y m o e a n p a r t / t i e s d e t e r m i n e d . The curvas were d r a w n from t he results of a slnljle e x p e r i m e n t a nd are representative of six performed.
627 then washed, incubated with opsonized zymosan and assayed for chemiluminescence. Superoxide dismutase or catalase completely protected the treated cell from damage (Fig. 8). In six experiments, superoxide dismutase protected 98 ± 4%, while catalase protected 95 + 6%. Although chemiluminescence was significantly higher with the catalase-incubated cells (Fig. 8), untreated neutrophils incubated with catalase alone, washed and exposed to opsonized zymosan also gave higher results (data n o t shown). Heat-inactivated enzymes did not protect the cells from damage. Neither the hydroxyl scavengers, mannitol, benzoate and ethanol [16], nor the singlet oxygen scavengers, tryptophan and histidine [17], were capable of inhibiting the oxidant-mediated damage. Discussion As leucocytes migrate into an inflammatory focus, resident phagocytes m a y provide a hostile extracellular oxidative environment. Exposure of polymorphonuclear neutrophils to an enzyme system capable of generating oxygen species similar to those generated by h u m a n phagocytes failedto impair the cells' ability to respond metabolically to a phagocytic load. Recently, it has been demonstrated that phagocytizing granulocytes rapidly deplete their intracellufar glutathione [8]. Thus, we have examined the abilityof polymorphonuclear neutrophils exposed to the potent thiol~xidizing agent diamide to withstand extracellular oxidant stress. Kosower et al. [10] has demonstrated that this compound reacts with glutathione more rapidly than other small molecular weight constituents. After combining rate data with concentration data, they concluded that glutathione was the major target for diarnide in the cell. This reagent has been used extensively for this purpose in a variety of biological systems [18--21]. Oliver et al. [15] demonstrated that diamide causes a rapid and reversible fall in soluble sulfllydryl compounds in the h u m a n granulocyte. Recently, Power et al. [22] reported that the incubation of rabbit polymorphonuclear neutrophils with diamide resulted in bleb formation and an inhibition of phagocytosis. In their study, the cells were incubated with 100 nmol of diamide/106 cells at 4°C for I0 rain before examination. At this temperature and concentration, the soluble sulfhydryls would be rapidly oxidized without cellular regeneration thus allowing diamide to react with greater degree of nonspecificity. The injury to granulocytes incubated simultaneously with diamide and xanthine-xanthine oxidase was inhibited by superoxide dismutase or catalase. Superoxide dismutase reduces the O~- concentration while increasing available H202 and catalase reduces H202 without affecting the concentration of O~-. Thus, the treated leucocyte is not harmed significantly by extracellular exposure to H~O2 or O~- alone. In order to explain similar observations in different systems, reaction schemes have involved an interaction between O~and H202. The OH" and O~ are compounds thought to be generated in such a manner [2,4,11], but in our system neither agent appeared responsible for the damage. Alternatively, hydroperoxides may be formed which have been reported to affect RBC membrane function [23] and impair alveolar macrophage phagocytosis [24]. At present, the exact reaction scheme responsible is unknown. Sagone et al. [25] have reported thtat lymphocyte function is ira-
628
paired by similar concentrations of xanthine-xanthine oxidase alone via a H20~dependent mechanism. Thus, it appears that lymphocytes and granulocytes have different sensitivities to oxidants and perhaps different mechanisms for protection. The actual site of damage is unknown. The cell appears unable to phagocytize but excludes trypan blue and responds to a second challenge of diamide. Since adherence of particles (via complement or immunoglobulin) alone m a y stimulate metabolism [26], it appears that membrane receptors or more distal cell function is impaired. Attempts to repair the cellwith reducing agents have thus far been unsuccessful. In a more complex system, Salin and McCord [4] demonstrated that the death of granulocytes 10--35 h after phagocytosis was inhibitable by superoxide dismutase, catalase and the OH" scavenger mannitol. In their system, the post-phagocytic cell m a y be rendered even more susceptible to oxidant stress such that the OH" m a y actually cause cell death. In addition, Clark and Klebanoff [27] have shown that the myeloperoxidase-H202halide system has a cytotoxic effect on intact leucocytes within 30--60 min. The susceptibility of cells with an altered thiol
BBA 28971
THE EFFECT OF OXIDANT STRESS ON DIAMIDE-TREATED HUMAN GRANULOCYTES
S T E P H E N J. WEISS * and A R T H U R
L. S A G O N E , Jr.
Division of Hematology and Oncology, Ohio State UniversityCollege of Medicine, Columbus, O H 43210 (U.S.A.) (Received November 16th, 1978)
Key words: Neutrophil; Diamide; Oxidant stress; Chemiluminescence; Phagocytosis; Superoxide dismutase
Summary The role of sulfhydryls in the protection of human polymorphonuclear neutrophfls against extraceUular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant~enerating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xanthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants. Introduction
Granulocytes generate a host of potentially toxic oxygen intermediates during phagocytosis [1]. These compounds have been implicated in the bac* T o w h o m c o ~ e s p o n d e n c e s h o u l d be addressed at: S i m p s o n M e m o r i a l R e s e a r c h I n s t i t u t e , 1 0 2 O b s e r v a t o r y Lane, A n n A r b o r , MI 4 1 8 0 9 , U . S . A .
621
tericidal mechanism, inflammation and tumoricidal capability [2--5]. Harmful compounds generated include superoxide anion (O~-), hydrogen peroxide (H202) and the products of their interaction, the hydroxyl radical (OH') and singlet oxygen (O~) [1]. During the act of ingestion, these reactive substances are released into the phagocytic vacuole as well as the extracellular milieu. In order to protect itself from oxidant injury, the granulocyte is armed with a variety of detoxifying mechanisms. Superoxide is reduced to H202 by the enxyme superoxide dismutase [6], while H202 may be degraded by catalase, myeloperoxidase or the glutathione~lutathione peroxidase system [6,7]. However, as granulocytes phagocytize, there is a p r o m p t and nearly complete depletion of intracellular glutathione [8]. Thus, the post-phagocytic granulocyte may be rendered more susceptible to oxidant injury. In this study, we evaluated the capacity of granulocytes treated with the thiol-oxidizing agent, diamide [ 9,10 ], to withstand extracellular oxidant exposure. Materials and Methods
Xanthine, xanthine oxidase ( 1 2 5 U / m l ) , bovine superoxide dismutase (3000 U/rag) catalase (type C40), diamide, mannitol, benzoate, L-tryptophan, zymosan, and L-histidine were obtained from the Sigma Chemical Co., St. Louis, MO. The catalase was freed of contaminating superoxide dismutase by repeated washings over an XM-100 Diaflo ultrafiltmtion membrane from the Amicon Corp., Scientific Systems Div., Lexington, MA [11]. Selegman's balanced salt solution, Earle's balanced salt solution and Hank's balanced salt solution were purchased from Grand Island Biological Co., Grand Island, NY. Cell preparation. Venous blood was collected in EDTA from normal healthy volunteers. Granulocytes were isolated by dextran sedimentation. The granulocytes were further purified by Ficoll-Hypaque gradient centrifugation to remove contaminating mononuclear cells [12]. The final preparations conrained greater than 98% granulocytes and less than 2% mononuclear cells. Exposure to 02 metabolites. In order to expose the cells to extracellular oxygen metabolites, 20 • 106 polymorphonuclear neutrophils were suspended in Selegrnan's balanced salt solution with 10 -4 M xanthine and 7.5 • 10 -3 U/ml of xanthine oxidase for 40 rain (unless otherwise indicated) in the presence or absence of 25 nmol diamide/106 cells at 37 ° C. At the end of the incubation period, the cells were diluted in ice-cold buffer, washed (400 X g, 10 min), resuspended in Hank's balanced salt solution, recounted and immediately assayed for metabolic parameters. In some experiments, the following agents were added during the incubations: 1.0/~g/rnl superoxide dismutase (3000 units/mg), 250/~g/ml catalase, 10 mM mannitol, 20 mM benzoate, 40 mM ethanol, 10 mM tryptophan or 10 mM histidine. Glucose metabolism. Hexose monophosphate shunt activity was estimated from the production of labeled CO2 from [1-14C]glucose [13]. The generation of 14CO2 by granulocyte suspensions was measured continuously using the ionization chamberelectrometer m e t h o d as previously described [13]. 1--2. 107 granulocytes were suspended in 4 rnl of Earle's balanced salt solution with 50 rag/100 ml of glucose and 5/~Ci of [1-~4C]giucose and added to 25-ml triple-headed distilling flasks. The inlet of the metabolic flask was connected to
622
a gas cylinder containing compressed air with 5% CO:. The outlet arm of the flask was connected to a Cary-Tolbert ionization chamber and a Cary Model 401 vibrating reed electrometer (Cary Instruments, Fairfield, NJ). The third arm of the flask was covered with a rubber stopper through which reagents could be added. The incubation flask were stirred continuously. After baseline 14CO2 production was established, opsonized zymosan particles were prepared by the method of Webb et al. [14] and added in 0.4 ml of normal saline (4 • 10 s particles/ml). In some experiments, the cells were stimulated by the addition of 25--50 nmol diamide/10 ~ cells or the xanthine.xanthine oxidase system. Results were expressed as mV generated/107 cells. Formate oxidation. H202 generation was estimated by the generation of labeled CO2 from [14C]formate [13]. The method employed was identical to the glucose metabolism experiments except that 5/~Ci (50 Ci/mol) of [14C]formate was added in place of labeled glucose. Results were expressed as mV generated/107 cells. Phagocytosis. Phagocytosis was estimated morphologically by counting 100 cells following incubation with zymosan particles at 37°C. Aliquots of cells were removed and examined at 5, 10, 30 and 60 rain after the addition of zymosan particles. Results were expressed as per cent of control phagocytosis at 60 rain unless otherwise indicated. Chemiluminescence assay. Chemiluminescence was studied using a Packard Model 3225 liquid scintillation counter (Packard Instrument Co., Inc., Downers Grove, IL), in a dark-room as described by Webb et al. [14]. 1--2 • 107 granulocytes were suspended in 6.6 ml of Earle's balanced salt solution in siliconized liquid scintillation vials. Zymosan was added in 0.4 ml of saline as in the other experiments. Results
Exposure of polymorphonuclear neutrophils to the oxygen metabolites generated by the xanthine-xanthine oxidase system [ 11] was associated with a stimulation of both [1.14C]glucose oxidation and [14C]formate oxidation which was maximal by 30 rain and then gradually decreased toward baseline (Fig. 1). Thus, it appears that polymorphonuclear neutrophils can degrade the oxidants generated outside the cell by both glutathione peroxidase and catalase and suggests these enzymes may afford protection against oxidant stress. In order to determine if the exposure of the polymorphonuclear neutrophils to extracellular oxidants incurred any metabolic damage, the ability of these cells to oxidize [14C]formate, [1-14C]glucose, chemiluminesce and phagocytize was studied. Granulocytes exposed to the xanthine-xanthine oxidase system for 40 rain, washed, resuspended and incubated with a phagocytic stimulus (zymosan) performed comparably to untreated cells (Table I). Increasing the xanthine-xanthine oxidase concentration ten fold did not alter the results. As previously reported, exposure of neutrophils to diamide caused a marked stimulation of [1.14C]glucose oxidation [15] (Fig. 2). Presumably this stimulation is mediated by oxidation of intracellular glutathione followed by its reduction by glutathione reductase and NADPH generated by the hexose monophosphate shunt [7]. Exposure of polymorphonuclear neutrophils to diamide alone
623 3r
Xanthine
Oxidase = | A~ded "~ 2 t- X-nthine I J" I Added I /
|
-60
0
I0
20 30 40 TIME { rains.)
50
60
70
F i g . 1. E f f e c t o f x a n t h i n e - x a n t h i n e o x i d a s e s y s t e m o n t h e o x i d a t i o n o f [ 1-I 4 C ] g l u c o s e a n d [ 14 C ] f o r m a t e oxidation by polymorphonuclear neutrophil. The curves represent a continuous measurement of 14CO2 production from [l-14C]glucose (c 9) o r [ 1 4 C ] f o r m a t e (e ~) and were d~awn from the data p o i n t o f a single e x p e r i m e n t . A f t e r s t e a d y - s t a t e c o n d i t i o n s w e r e e s t a b l i s h e d , 1 0 -4 M x a n t h i n e w a s a d d e d f o l l o w e d b y t h e addition o f 9 . S • 1 0 - 3 U / m l o f x a n t h i n e o x i d a s e . T h e y - a x i s i n d i c a t e s t h e e l e c t r i c a l s i g n a l in m V g e n e r a t e d b y 1 4 C O 2 / 1 0 9 cells. T h e e x p e r i m e n t is t y p i c a l o f f o u r p e r f o r m e d .
did not stimulate [6-t4C]glucose oxidation, [14C]formate oxidation or chemiluminescence. Neutrophils incubated with diamide for 40 rain, washed and allowed to phagocytize performed comparably [1-14C]glucose, [14C]formate, chemiluminescence and phagocytosis) to untreated cells.Thus, it appeared that exposure of polymorphonuclear neutrophils to xanthine-xanthine oxidase or diamide alone did not impair the metabolic response associated with phagocytosis. In order to study the response of thiol~iepleted cells to withstand extracellular oxidants, granulocytes were simultaneously exposed to diamide and the xanthine-xanthine oxidase system for 40rain, washed, resuspended and allowed to phagocytize. The chemiluminescence response of these treated washed cells in response to opsonized zymosan was markedly impaired (Fig. 3). In eleven experiments, peak chemiluminescence was impaired 80 + 8%. The degree of impairment was time dependent, i.e. the greater the incubation period with the xanthine-xanthine oxidase system the greater the injury (Fig. 4). It was always necessary to incubate the cells simultaneously with the enzyme system and diamide in order to demonstrate the maximal impairment of the polymorphonuclear neutrophil. The ability of the cells to oxidize [1J4C]glucose or [14C]formate during phagocytosis was also significantly impaired (Figs. 5 and 6). In four experiments [14C]formate and [1J4C]glucose oxidation were inhibited 72 + 8% and 83 -+ 7%, respectively.
TABLE I METABOLIC STATUS OF XANTHINE-XANTHINE
OXIDASE-TREATED
GRANULOCYTES
A s s a y p e r f o r m e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . R e s u l t s e x p r e s s e d as m e a n ± S . D . o f f o u r e x p e r i ments. Assay
% of control
[1-14C]Glueose oxidation [14C]Formate oxidation Chemiluminescence Phagocytolis
96 100 102 98
± ~ + ±
5 4 3 4
624
2 ¸
i
diamide
II -60
I
,
0
l, 15
,
, I
I
i
I
I
I
30
45
60
75
90
105
time (minutes) Fig. 2. E f f e c t of d i a m i d e o n t h e o x i d a t i o n o f [ l - 1 4 C ] g l u c o s e b y p o l y m o r p h o n u c l e a r n e u t r o p h i l . The c u r v e s r e p r e s e n t a c o n t h l u o u s m e a s u r e m e n t o f 1 4 C O 2 p r o d u c t i o n f r o m [1 -I 4 C ] g l u e o s e a n d w e r e d r a w n f r o m t h e d a t a p o i n t o f a single e x p e r i m e n t . A f t e r s t e a d y - s t a t e c o n d i t i o n s were e s t a b l i s h e d , d i a m i d e (25 n m o l / 1 0 6 p o l y m o r p h o n u c l e a r n e u t z o p h i l ) was a d d e d t o o n e s u s p e n s i o n (o o ) as i n d i c a t e d b y t h e a r r o w , The s e c o n d s u s p e n s i o n (e -') w a s u s e d as t h e c o n t r o l . The y-axis i n d i c a t e s t h e e l e c t r l c a l signal i n m V g e n e r a t e d b y 1 4 C O 2 / 1 0 7 cells, The e x p e r i m e n t is t y p i c a l o f e i g h t p e r f o r m e d .
Although the metabolic response of the treated cells was significantly impaired, viability was judged greater than 95% by Trypan Blue exclusion. Examination of these cells after incubation with opsonized zymosan revealed
12
101
I0 w
~o 8 w
i6 4
21 I 7
I 14
I 21
I 28
I
I
35
7
I
/
I
l
14 21 28 35 time (minutes) time (minutes) Fig. 3. E f f e c t o f x a n t h i n e - x a n t h i n e o x i d a s e or x a n t h i n e - x a n t h i n e o x i d a s e plus d i a m i d e o n p o l y m o r p h o n u c l e a r n e u t ~ o p h i l c h e m i l u m i n e s c e n c e . P o l y m o r p h o n u c l e a r n e u t r o p h i l s w e r e i n c u b a t e d in b u f f e r a l o n e ( o - - - - - - . - ~ ) ; x a n t h i n e - x a n t h i n e o x i d a s e (a -') or x a n t h i n e - x a n t h i n e o x i d a s e and d i a m i d e ( i a) for 4 0 m i n , w a s h e d a n d t h e n r e s u s p e n d e d i n fresh buffer. The c h e m i l u m i n e s c e n c e g e n e r a t e d b y t h e p o l Y m o r p h o n u e l e a r n e u t r o p h i l s ( e p m / 1 0 6 ) d u r i n l t h e p h a g o c y t o s i s o f z y m o s e n p a r t i c l e s was t h e n d e t e r m i n e d . E a c h p o i n t is t h e m e a n o f t w o o b s e r v a t i o n s . The e x p e r l m e n t is t y p i c a l of 11 p e r f o r m e d . Fig. 4. E f f e c t o f t h e t i m e o f p r e i n c u b a t / o n w i t h the x a n t h i n e - x a n t h i n e o x i d a s e s y s t e m a n d d i a m i d e o n polymorphonuclear neutrophil chemiluminescence. Polymorphonuclear neutrophils were preincubated w i t h t h e x a n t h i n e - x a n t h i n e o x i d a s e s y s t e m a n d d i a m i d e for 1 5 r a i n ( ! -'), 3 0 r a i n (e -') or 4 5 rain (o ~), w a s h e d a n d t h e n r e s u s p e n d e d in fresh m e d i u m . The c h e m i l u m i n e s c e n c e o f t h e p o l y m o r p h o n u e l e a x n e u t r o p h i ] s ( c p m / 1 0 6 ) duldng the p h a g o c y t o s i s o f opsondzed z y m o s a n p a r t i c l e s w a s t h e n d e t e r m i n e d . The results r e p r e s e n t s a single e x p e r i m e n t w i t h is t y p i c a l of f o u r p e r f o r m e d . Ae i n d i c a t e d , ceils i n c u b a t e d w i t h x a n t h i n e - x a n t h i n e o x l d a s c a l o n e (o o) w e r e n o t i m p a i r e d (see Fig. 3).
625
12
~ 4
-
60
zymo~n added
0
10
20
30
40
50
60
time (minutes)
Fig. 5. Effect of x a n t h i n e - x a n t h i n e oxidase or x a n t h i n e - x a n t h i n e oxidase plus di a mi de on p o l y m o r p h o nuclear neutxophil hexose m o n o p h o s p h a t e shunt activity. P o l y m o r p h o n u c l e a r n e u t r o p h i l s were prelneub a t e d with x a n t h i n e - x a n t h i n e oxidase alone ( o - - - - - - - ~ ) or x a n t h i n e - x a n t h i n e oxidase plus di a mi de for 40 m i n ( e - --), washed and resusPended in fresh m e d i u m in a m e t a b o l i c flask w i t h [ l - 1 4 C ] g l u c o s e (see Materials and Methods). Curves represent a c o n t i n u o u s meamarement of 14CO2 p r o d u c t i o n from [ l - 1 4 c ] g l u c o s e . The y-axis indicates the electrical 'signal in m V generated by t he 14CO2. After steadystate c o n d i t i o n s were established, Opsonized z y m o s a n particles were a dde d as i ndi c a t e d b y t he arrows. The curves were d r a w n from the d a t a p o i n t of a single e x p e r i m e n t and are representative of four performed.
greater than 90% inhibition of phagocytosis. Metabolism studies with opsonized bacteria or latex particles were similar to experiments performed with zymosan. The ability o f the diamide-xanthine-xanthine oxidase-treated granulocytes to respond to a non-phagocytic stimulus of metabolism was studied by incubating the cell with a second dose of diamide and then measuring [1-14C]glucose oxidation (Fig. 7). In three experiments, [1-14C]glucose oxidation by the injured cells was 56 + 8% o f controls.
2.0
u
~ 1.0 O.5
0
10
20
30
40
50
60
time (minutes)
Fig. 6. Effect of x a n t h l n e - x a n t h i n e oxidase or x a n t h i n e - x a n t h i n e oxidase plus d i a m i d e on p o l y m o r p h o nuclear n e u t r o p h i l formate o x i d a t i o n . P o l y m o ~ h o n u e l e a r n e u t r o p h i l s were i n c u b a t e d w i t h xa nt hi ne x a n t h i n e oxidase alone (oo) or xaDthine-xanthine oxidase pl us d i a m i d e for 40 m l n (~~), washed and resuspended in fresh m e d i u m in a m e t a b o l i c flask w i t h [ 1 4 C ] f o n n a t e (see Materials and Methods). The eurves represent a c o n t i n u o u s m e a s u r e m e n t of 14CO2 p r o d u c t / o n from [ 1 4 C ] f o r m a t e , The y ~ indicates the electrical signal in m V generated b y t h e 14CO2. A ft e r stcady-state c o n d i t i o n s were establ/shed, opson/zed z y m o R n partleles were added as i n d i c a t e d b y t he arrows. The curves were d r a w n from the d a t a p o i n t s of a single e x p e r i m e n t and are representative of four pe rforme d.
626
i0I i"l:m./f "6()
0
10
20 30 tlme (minutes)
40
50
60
l~Ig. 7. Effect of o x i d a n t injury on the d/amide response of p o l y m o r p h o n u c l e a r neutrophfls, o . o, the o x i d a t i o n of [1-14C]illueose by p o l y m o r p h o n u c l e ~ neut~oph~lls p r e i n c u b a t e d w i t h xanthineoxanthine ox/dase, and • --, p r e t n ~ b a t e d w i t h xant~dne-xanthine oxidase and diamide. After steady-state condiflons were established, 50 n m o l / 1 0 6 ceils of d i a m k l e was added a s / n d i c a t e d b y t he arrows. The curves were d r a w n fro m th e d a t a p o / n t of a single e x p e r i m e n t and are representative of three performed.
In an attempt to determine the specific oxygen intermediate responsible for the damage, diamide-treated cells were incubated with various scavengers during their exposure to the xanthine-xanthine oxidase system. The cells were 16
14
12
0
7
14
21
28
35
time (minutes) F/g. 8. lhroteetive effect o f s u p m x i d e d i ~ ; u t a s e and c a t a l a n on the o x i d a n t inJm'y produced b y xantbtne- xan thine oxldmm ~ e s y s t e m . P o l y m o r p h o n u d e a r neutrophfls were I)reinoubated w i t h x a n t b l n e x a n t h l n e ox/dase (¢ --); x a n t h i n e - x a n t h i n e oxidase plu s d/am/de (s..-------.a); x a n t h i n e - x a n t h i n e oxidase, diamid e and s u p e r o x l d e d/mnutaas (o------.--¢); or x a n t h / n e - x a n t h / n e ox/dmm, d/m~ide and catalase (c~---------~). The cells were t h a n washed and the chemllumtnesolmce generated d u r l n l t he pha goc yt os / s of z y m o e a n p a r t / t i e s d e t e r m i n e d . The curvas were d r a w n from t he results of a slnljle e x p e r i m e n t a nd are representative of six performed.
627 then washed, incubated with opsonized zymosan and assayed for chemiluminescence. Superoxide dismutase or catalase completely protected the treated cell from damage (Fig. 8). In six experiments, superoxide dismutase protected 98 ± 4%, while catalase protected 95 + 6%. Although chemiluminescence was significantly higher with the catalase-incubated cells (Fig. 8), untreated neutrophils incubated with catalase alone, washed and exposed to opsonized zymosan also gave higher results (data n o t shown). Heat-inactivated enzymes did not protect the cells from damage. Neither the hydroxyl scavengers, mannitol, benzoate and ethanol [16], nor the singlet oxygen scavengers, tryptophan and histidine [17], were capable of inhibiting the oxidant-mediated damage. Discussion As leucocytes migrate into an inflammatory focus, resident phagocytes m a y provide a hostile extracellular oxidative environment. Exposure of polymorphonuclear neutrophils to an enzyme system capable of generating oxygen species similar to those generated by h u m a n phagocytes failedto impair the cells' ability to respond metabolically to a phagocytic load. Recently, it has been demonstrated that phagocytizing granulocytes rapidly deplete their intracellufar glutathione [8]. Thus, we have examined the abilityof polymorphonuclear neutrophils exposed to the potent thiol~xidizing agent diamide to withstand extracellular oxidant stress. Kosower et al. [10] has demonstrated that this compound reacts with glutathione more rapidly than other small molecular weight constituents. After combining rate data with concentration data, they concluded that glutathione was the major target for diarnide in the cell. This reagent has been used extensively for this purpose in a variety of biological systems [18--21]. Oliver et al. [15] demonstrated that diamide causes a rapid and reversible fall in soluble sulfllydryl compounds in the h u m a n granulocyte. Recently, Power et al. [22] reported that the incubation of rabbit polymorphonuclear neutrophils with diamide resulted in bleb formation and an inhibition of phagocytosis. In their study, the cells were incubated with 100 nmol of diamide/106 cells at 4°C for I0 rain before examination. At this temperature and concentration, the soluble sulfhydryls would be rapidly oxidized without cellular regeneration thus allowing diamide to react with greater degree of nonspecificity. The injury to granulocytes incubated simultaneously with diamide and xanthine-xanthine oxidase was inhibited by superoxide dismutase or catalase. Superoxide dismutase reduces the O~- concentration while increasing available H202 and catalase reduces H202 without affecting the concentration of O~-. Thus, the treated leucocyte is not harmed significantly by extracellular exposure to H~O2 or O~- alone. In order to explain similar observations in different systems, reaction schemes have involved an interaction between O~and H202. The OH" and O~ are compounds thought to be generated in such a manner [2,4,11], but in our system neither agent appeared responsible for the damage. Alternatively, hydroperoxides may be formed which have been reported to affect RBC membrane function [23] and impair alveolar macrophage phagocytosis [24]. At present, the exact reaction scheme responsible is unknown. Sagone et al. [25] have reported thtat lymphocyte function is ira-
628
paired by similar concentrations of xanthine-xanthine oxidase alone via a H20~dependent mechanism. Thus, it appears that lymphocytes and granulocytes have different sensitivities to oxidants and perhaps different mechanisms for protection. The actual site of damage is unknown. The cell appears unable to phagocytize but excludes trypan blue and responds to a second challenge of diamide. Since adherence of particles (via complement or immunoglobulin) alone m a y stimulate metabolism [26], it appears that membrane receptors or more distal cell function is impaired. Attempts to repair the cellwith reducing agents have thus far been unsuccessful. In a more complex system, Salin and McCord [4] demonstrated that the death of granulocytes 10--35 h after phagocytosis was inhibitable by superoxide dismutase, catalase and the OH" scavenger mannitol. In their system, the post-phagocytic cell m a y be rendered even more susceptible to oxidant stress such that the OH" m a y actually cause cell death. In addition, Clark and Klebanoff [27] have shown that the myeloperoxidase-H202halide system has a cytotoxic effect on intact leucocytes within 30--60 min. The susceptibility of cells with an altered thiol
