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The role of the phagocyte in host-parasite interactions
168
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25912 T H E R O L E OF T H E P H A G O C Y T E IN H O S T - P A R A S I T E I N T E R A C T I O N S X l I I . T H E D I R E C T Q U A N T I T A T I V E ESTIMATION OF H 2 0 2 IN P H A G O C Y T I Z I N G CELLS B. P A U L AND A. J. S B A R R A
Department of Pathology and Medical Research, St. Margaret's Hospital and Department of Obstetrics and Gynecology, Tufts University School of Medicine, Boston, Mass. (U.S.A .) (Received July 3ISt, 1967) (Revised manuscript received October 23rd, 1967)
SUMMARY
I. A technique that allows the direct quantitative estimation of H=O 2 in polymorphonuclear leukocytes has been developed. 2. Lysis or homogenization followed by dialysis of polymorphonuclear leukocytes results in a dialysate containing fluorescence-producing material. 3- This material is catalase sensitive. 4- Catalase specificity cannot be demonstrated without preliminary dialysis, indicating the presence of a non-dialyzable catalase-limiting component(s) in the polymorphonuclear leukocytes. 5- Phagocytizing cells contain 2-4 times as much measurable H202 as nonphagocytizing polymorphonuclear leukocytes.
INTRODUCTION
Stimulated metabolic events associated with phagocytizing cells have been well established 1-5. Increased glycolysis, oxygen consumption, oxidation of glucose through the hexose monophosphate pathway and increased formate oxidation, are major metabolic events associated with phagocytosis. In an attempt to delineate the mechanism(s) responsible for the stimulated oxidative activities, a postulation that Hz02 is a functional end-product of this oxidative metabolism was entertained 4. The H 2 0 2 would arise from the oxidation of NADPH, produced from the hexose monophosphatc pathway, b y molecular oxygen. ZATTI AND ROSSI6 have provided convincing evidence that a granule-bound NADPH-oxidase is critical in stimulating the cyanide-insensitive oxygen uptake noted during phagocytosis, and that during the process N A D P H is oxidized. BECK7 reported that NADP + is a rate-limiting factor for the hexose monophosphate pathway in human leukocytes and it has been noted in our laboratory that the addition of NADP + to guinea pig leukocyte homogenates stimulated the hexose monophosphate pathway 5. An increase in N A D P + / N A D P H ratios in phagocytizing cells has been reported by ZATTI AND ROSSI8 and by our laboratory 9. IYER AND QUASTEL 1° have Biochim. Biophys. Acta, 156 (1968) 168 178
ESTIMATION OF
H~02 IN PHAGOCYTES
169
demonstrated an enzyme system in guinea pig leukocyte homogenates capable of oxidizing N A D P H and to a lesser extent NADH, b y molecular oxygen. The increased formate oxidation exhibited b y phagocytizing cells led IYER, ISLAM AND QUASTEL 4 to predict increased H20 2 formation in these cells. It is known that formate oxidation is enhanced in the presence of H~O 2 and catalase 11. All of the above data provide indirect evidence that phagocytosis is accompanied b y an increase in H,O 2 production. The bactericidal nature of H20 2 has long been known TM. However, to the best of our knowledge a direct quantitative measure of the H,O 2 concentration in phagocytes has not been reported, Our recent finding that polymorphonuclear leukocytes are more efficient in killing Escherichia coli aerobically (supposedly when H20 2 formation is optimal) than anaerobically 5, suggested a study designed to measure directly the H20 2 concentration in resting and phagocytizing cells. This report will present the results obtained from such a study. MATERIAL AND METHODS
Reagents. All chemicals used were of reagent grade or better. Peroxidase (Horseradish) Type I I was purchased from the Sigma Chemical Co., St. Louis, Mo. and contained 127.o Purpurogallin units per mg of protein. Purified rat liver catalase was a gift from Dr. E. Kuchinskas of the State University of New York, Brooklyn, N.Y. Merck hydrogen peroxide (30 %) reagent grade was used throughout. The fluorescence-producing compound leucodiacetyl-2,7-dichlorofluorescein was synthesized according to the method of BRANDT AND KESTON13. Preparation of reagents. (I) H20 2 solutions were prepared daily by diluting 30 % H20 2 2o00 × with distilled water. The 230 m/~ absorbance of this solution was recorded. The concentration was calculated from the extinction coefficient of 81 cm -1 mole -1 at 230 m/~ and further dilutions were made in distilled H20 (ref. 14). (2) Catalase solutions were prepared in 0.025 M phosphate buffer 15 at p H 7.0. The enzyme assayed in our laboratory had the following specifications. Activity, 43 IOO units]mg protein. Protein content, 13.o mg/ml based on 407 m~ A and 430000 molar extinction coefficient. The 407/280 A ratio was o.91. In all cases I unit of catalase equals I/~mole of H20 2 decomposed/min at p H 7.0 and 25 °, assayed as described by B E E R S AND SIZER 14.
(3) Peroxidase solutions were made to i mg/ml in 0.06 M phosphate buffer at p H 6.0. (4) A stock solution of leucodiacetyl-2,7-dichlorofluorescein was made to lO-4 M in ethanol and stored at 4 °. Fluorometric determination of H202. The method of KESTON AND BRANDT16 with modifications was employed for the fluorometric assay of H,Oz in the m/, molar range. The reaction mixture, in a total of 5.0 ml contained in m~moles; phosphate buffer, I.O. lO 5 (pH 6.0) ; ZnSO 4. 7 H202, 230; leucodiacetyl-2,7-dichlorofluorescein, 8.0; H20 2 or samples from polymorphonuclear leukocytes containing HzO ~ in the range of o.o4-1.6; and peroxidase, 38 Purpurogallin units. Peroxidase was added last to start the reaction, in the absence of added horseradish peroxidase the reaction would not start, and after 2o min of incubation at room temperature, readings were recorded. Fluorometrie assays were carried out in a Beckman DU spectrophotometer with a fluorescence attachment No. 73500. For hydrogen peroxide absorbance and Biochim. Biophys. Acta, 156 (I968) I 6 8 - 1 7 8
17o
B. PAUL, A. J. SBARRA
catalase assay, a Beckman model DB speetrophotometer was used. Hydrogen peroxide and catalase solutions gave the same absorbances in both instruments. Preparation ofpolymorphonuclear neutrophils. Guinea pigs of both sexes weighing 300-400 g were used throughout. Peritoneal exudates were prepared by a previously published procedure e. The exudate containing over 9 ° % polymorphonuclear leukocytes was washed 3 times in Ca-free Krebs-Ringer phosphate medium 4 and centrifuged at 400 × g for 4 rain. The pellet was brought up to a desired volume and the absorbance at 600 mr* was determined in a Spectronic 20, Bausch and Lomb colorimeter. An A of I.O was found to be equivalent to 172/zg cellulai phosphorus (IOO/zg phosphorus is equal to 5.5" lO7 polymorphonuclear leukocytes). Test particles. IS-h cultures of E. coli and Staphylococcus albus grown in trypticase soy broth served as test particles. Before use they were autoclaved for 30 min at 121 °. The resulting heat-killed bacteria were washed twice and suspended in Krebs-Ringer phosphate medium. Polystyrene latex spherules, o . 8 I / , in diameter were purchased from the Difco Laboratories, Detroit, Mich. Lysis and dialysis of polymorphonuclear leukocytes. 4 vol. of distilled H20 (pH 5.o), were added to a polymorphonuclear leukocyte pellet (containing 50 /zg cellular phosphorus per ml) held in an ice-bath. After io rain the lysed cells were centrifuged at 12o00 × g for 20 rain. An aliquot of the supernatant was assayed for H202 directly, another aliquot was dialyzed in an ice-bath for 30 min against H20 (pH 5.o), and the dialysate assayed. Similarly, polymorphonuclear leukocytes held in Krebs-Ringer phosphate medium and dialyzed against 15-4o vol. of KrebsRinger phosphate medium were treated and examined as above. During dialysis, the polymorphonuclear leukocyte preparations were continuously agitated by means of rotating circular shaker (115 rev./min).
Estimation of total HeO 2 in polymorphonuclear leukocytes and polymorphonuclear leukocyte homogenates. Since the dialysis of polymorphonuclear leukocytes yielded dialysates suitable for H202 estimation, it was felt that studies concerned with recovery of added H202 after dialysis would be required; 12. 4 and 12.7 m/zmoles of HeO 2 in I.O ml of Krebs-Ringer phosphate medium and/ol H20 (pH 5.o), were dialyzed against 15 ml of Krebs-Ringer phosphate medium or H20 (pH 5.0) for various periods of time. For controls the same H202 concentrations were added to 15 ml of Krebs-Ringer phosphate medium. The dialysates and controls (not dialyzed) were assayed fluorimetrically. Having established optimal conditions for H 2 0 2 recovery, polymorphonuclear leukocytes samples were now dialyzed in Krebs-Ringer phosphate medium and in H20 (pH 5.o), for different periods of time and the dialysates assayed for H20 2. All dialyses were carried out at o °. Polymorphonuclear leukocytes in Krebs-Ringer phosphate medium were homogenized for 5 min using a motor-driven pestle with a teflon tip (disruption of cells was confirmed by light microscopy). An aliquot of the homogenate was dialyzed in Krebs-Ringer phosphate medium and also in distilled H~O (pH 5.o), for different periods of time; also an aliquot was added directly to Krebs-Ringer phosphate medium and to H20 (pH 5.o), and after 30 min each was centrifuged at 17300 × g for 30 min. Intact polymorphonuclear leukocytes were tested as above. All dialysates and supernatants (lysates) were assayed for H202.
Estimation of H202 production by polymorphonuclear leukocytes in the absence Biochim. Biophys. Acta, 156 (1968) 168-178
ESTIMATION OF H20 2 IN PHAGOCYTES
171
and presence of particles. Experiments designed to detect the total H , O , content of polymorphonuclear leukocytes at rest and during phagocytosis were attempted. Polymorphonuclear leukocytes in the absence and presence of particles were incubated in polyethylene tubes and after different periods of time, supernatants were assayed for H20 2. The results obtained indicated an increased H20 2 content in phagocytizing cells, however, they were not consistent. This was probably due to the complex nature of the phagocytic system (i.e. catalase, myeloperoxidase, and interfering substances present in the polymorphonuclear leukocyte preparation). In order to circumvent this problem, phagocytosis was allowed to occur in 3 different dialysis bags. One was held at o ° and served to establish the endogenous H20 2 content. The second and third bags contained polymorphonuclear leukocytes with and without different particles and they were both incubated at 37 ° . Heatkilled E. coli, S. albus and polystyrene latex spherules served as test particles. The resulting dialysates were assayed for H20 2. RESULTS
The fluorescence obtained when known concentrations of H,O 2 were assayed are plotted in Fig. I. A linear relationship exists at low HzO 2 concentrations and this part of the curve was always used for the analyses.
0 0
20
40
60
~ , mpmoies x 1110
80
i00
Fig. I. Relationship between fluorescence and hydrogen peroxide concentrations. 30% H202 (Merck) diluted with distilled H~O. Fluorescence measured after 20 min incubation at 25 °. Fluorescence plotted against re#moles of H202 contained in 5 ml of the assay mixture.
Direct measurement of H~O 2 in polymorphonuclear leukocytes lysates was found to be satisfactory, but the observed fluorescence was not destroyed b y catalase and the addition of H202 to the lysate did not increase fluorescence. It would appear that interfering substances are present in polymorphonuclearleukocyte lysates. These results m a y be seen in Table I. In an attempt to circumvent the problem of interfering material in polymorphonuclear leukocytes, dialysis of polymorphonuclear leukocytes under different experimental conditions was tried. Fluorescence was noted in the dialysate and further Biochim. Biophys. dora, 156 (i968) 168-178
I72
B. PAUL, A. J. SBARRA
TABLE I FLUORESCENCE
PRODUCED
BY POLYMORPHONUCLEAR
Sample
H202, re#moles*
Lysate * * Lysate -I- catalase § Lysate + HlO~ §§ Lysate + H~O 2 + catalase
6.59 7.38 6.38 6.95
LEUKOCYTE
LYSATE
~ 0.64*** ± 0.60 ± 0.59 4:0.45
All results adjusted for IOO/zg cellular phosphorus or 5.5" lO7 cells. ** Io min after addition of 4 vol. of H20, pH 5.o at o °, the lysed cells were centrifuged at i2 ooo × g for 2o min and the supernatant (lysate) assayed. Readings were taken after a 2o min reaction time. Lysate from I-iO/2g cellular phosphorus per assay mixture. *** Mean ~- standard error of the mean of 3 experiments are presented in each case. § 0.75 unit of rat liver catalase per assay mixture. 15 nfin after addition of catalase (at 25 °) samples were assayed. §§ o.I6 or 0.32 m/~mole of H~O 2 (Merck) per assay mixture. See text for additional details. T A B L E II FLUORESCENCE PRODUCED BY POLYMORPHONUCLEAR LEUKOCYTE DIALYSATE
Treatment
H202, m~moles*
A. Polymorphonuclear leukocytes dialysed in Krebs-Ringer phosphate medium for 3 ° min. Dialysate assayed
1.56 ± o. I3 (I4)**
B. Polymorphonuclear leukocytes lysed with H20, pH 5.0. Supernatant of the lysate***: i. Dialysed in H20 (pH 5.o) for 3 ° min and dialysate assayed 2. Assayed directly
4.84 ~: 0.62 7.46 =t= 0.22
(6) (9)
* Dialysate or lysate from i - i o / ~ g cellular phosphorus assayed in each case and results adjusted for ioo/zg cellular phosphorus. ** Mean and standard error of the mean given in each case. The number in parentheses indicates the number of experiments performed. *** io min after addition of 4 vol. of H20 (pH 5.0) the lysed cells were centrifuged at 12 ooo × g for 20 rain and the supernatant was treated as indicated. Readings taken after 20 min reaction time. See Table I and text for additional details. i t w a s n o t e d t h a t l y s e d cells a f t e r d i a l y s i s s h o w e d g r e a t e r f l u o r e s c e n c e t h a n n o n - l y s e d cells. T h e s e r e s u l t s a r e s h o w n i n T a b l e I I . I n o r d e r t o a s c e r t a i n if t h e f l u o r e s c e n c e n o t e d i n t h e d i a l y s a t e s w a s d u e t o H20~ and not to a non-specific material, an attempt to destroy the fluorescent-producing material with catalase was studied. It can be seen in Table III that the catalasetreated dialysates showed essentially no fluorescence. This would appear to indicate t h a t t h e f l u o r e s c e n c e n o t e d i n t h e d i a l y s a t e s w a s d u e t o H 2 0 ~. T h e a d d i t i o n of 0.75 u n i t of t h e r a t l i v e r c a t a l a s e p e r a s s a y m i x t u r e w a s f o u n d n o t t o i n t e r f e r e w i t h t h e a s s a y s y s t e m w h i l e a t t h e s a m e t i m e i t w a s c a p a b l e of d e s t r o y i n g t h e f l u o r e s c e n c e p r o d u c i n g m a t e r i a l . S a m p l e s w e r e a s s a y e d a t 25 o, 15 m i n a f t e r t h e a d d i t i o n of c a t a l a s e . T o e s t a b l i s h f u r t h e r o p t i m a l c o n d i t i o n s for a s s a y , H 2 0 , r e c o v e r y e x p e r i m e n t s w e r e c a r r i e d o u t . I t c a n b e s e e n i n Fig. 2 t h a t i n 60 r a i n 90 % of t h e H 2 0 2 a d d e d t o a d i a l y s i s b a g is r e c o v e r e d i n t h e d i a l y s a t e . T h e effect of t h e t i m e of d i a l y s i s o n H 2 0 2
Biochim. Biophys. Acta, 156 (1968) i 6 8 - i 7 8
173
ESTIMATION OF H 2 0 2 IN PHAGOCYTES TABLE III DESTRUCTION LEUKOCYTE
OF
THE
DIALYSATES
FLUORESCENCE-PRODUCING BY
COMPONENT
(H202) OF
POLYMORPHONUCLEAR
CATALASE
Samples
HzO~, re#moles*
A. I. 2. 3. 4.
Krebs-Ringer Krebs-Ringer Krebs-Ringer Krebs-Ringer
B. I. 2. 3. 4.
Dialysate Dialysate Dialysate Dialysate
phosphate phosphate phosphate phosphate
medium medium medium medium
dialysates 1.63 ± o.3** (8) 0.02 (8) d i a l y s a t e s + c a t a l a s e * ** dialysates + HzO 2§ 97 % H202 recovered (3) d i a l y s a t e s + H202 + catalase o.o (3)
of l y s a t e of l y s a t e + catalase of l y s a t e + H202 of l y s a t e + H202 -[- catalase
3.8 ± 0.06 (8) 0.20 (3) 82 % H 2 0 ~ recovered (3) 5-7% H2Oi recovered (3)
(A) p o l y m o r p h o n u c l e a r l e u k o c y t e s d i a l y s e d in K r e b s - R i n g e r p h o s p h a t e m e d i u m for 3 ° m i n a t o °. D i a l y s a t e s a n a l y s e d as indicated. (B) p o l y m o r p h o n u c l e a r l e u k o c y t e s l y s e d w i t h H 2 0 (pH 5.0) (as in T a b l e I**). S u p e r n a t a n t dialysed for 30 rain a t o ° a n d a n a l y s e d as indicated. * R e s u l t s a d j u s t e d for ioo # g cellular p h o s p h o r u s . ** M e a n a n d s t a n d a r d error of t h e m e a n g i v e n in each case. N u m b e r in p a r e n t h e s e s refers to t h e n u m b e r of e x p e r i m e n t s . *** 0.75 u n i t of t h e r a t liver catalase per a s s a y m i x t u r e . 15 m i n after a d d i t i o n of c a t a l a s e (at 25 °) s a m p l e s were assayed. § o.16 a n d 0.32 m # m o l e of H 2 0 ~ (Merck) per a s s a y m i x t u r e . See Table I a n d t e x t for a d d i t i o n a l details. 100
IlO
.
~
60
M
100
DIALYSIS TIME, MINUTES
Fig. 2. R e c o v e r y of a d d e d h y d r o g e n peroxide (Merck) f r o m d i a l y s a t e s . 12. 4 a n d 12. 7 m/zmoles of H202 c o n t a i n e d in I.O m l of K r e b s - R i n g e r p h o s p h a t e m e d i u m , dialyzed a g a i n s t 15.o m l of K r e b s R i n g e r p h o s p h a t e m e d i u m . F o r controls t h e s a m e c o n c e n t r a t i o n s of H~O 2 directly a d d e d to 15.0 ml of K r e b s - R i n g e r p h o s p h a t e m e d i u m . T h e d i a l y s a t e s a n d controls a s s a y e d fluorometrically a n d r e s u l t s are p r e s e n t e d as p e r c e n t of control.
release by polymorphonuclear leukocytes after dialysis in Krebs-Ringer phosphate medium and in H20 (pH 5.0) is shown in Table IV. It is evident that lysed cells, (H20, pH 5.0) release more H20 2 than cells held in Krebs-Ringer phosphate medium, where the cells remain intact. With lysed ceils maximal release of H~O2 (dialysis) was achieved in 9° min. This time period was used in all subsequent experiments. Biochim. Biophys. Acta, 156 (1968) 168-178
174
B. PAUL, A. J. SBARRA
T A B L E IV THE
]~FFECT OF TIME
Dialysis time (min)
IO 20 3° 45 60 90
OF DIALYSIS
ON
H202
RELEASE
BY POLYMORPHONUCLEAR
LEUKOCYTES
H202, re#moles* dialysed in Krebs-Ringer phosphate medium 1.9 2. 3 2. 7 2. 9 3.6 3.9
(5)** (8) (8) (8) (8) (5)
H20 (pH 5.0)
3 .2 (4) 4 .0 (5) 5.4 (5) 9.5 (II) 11. 4 (IO)
* R e s u l t s a d j u s t e d for IOO/~g cellular p h o s p h o r u s . ** Mean a n d n u m b e r of e x p e r i m e n t s (in p a r e n t h e s e s ) g i v e n in each case. See Table I a n d t e x t for a d d i t i o n a l details.
TABLE V TOTAL MEASURABLE H202 LEUKOCYTE HOMOGENATES
OF
POLYMORPHONUCLEAR
Treatment
LEUKOCYTES
Polymorphonuclear leukocytes
AND
POLYMORPHONUCLEAR
Homogenate
H202 ml~moles/Ioo ttg cellular phosphorus Dialysis in K r e b s - R i n g e r p h o s p h a t e m e d i u m * Dialysis in H 2 0 (pH 5.o) Direct addition to H 2 0 (pH 5.0)***
3.90 (5)** ~3.o (8) I5.54 (4)
12.4 (5) 14.4 (8) 15.6o (6)
* Dialysis in all cases were carried o u t a t o ° for 9o min. D i a l y s a t e s assayed. ** M e a n a n d n u m b e r of e x p e r i m e n t s (in parentheses) g i v e n in each case. *** Direct a d d i t i o n to 15 vol. of H~O (pI-t 5.0). 3 ° rain after a d d i t i o n a n d at o °, c e n t r i f u g e d in t h e cold at 17 300 × g for 30 min. S u p e r n a t a n t s assayed. See Table I a n d t e x t for a d d i t i o n a l details.
An attempt was made to estimate the total measurable fluorescence-producing material (H202) of polymorphonuclear leukocytes and polymorphonuclear leukocyte homogenates. These results are presented in Table V. It appears that no significant differences are evident between the different experimental conditions. It should be noted that approx. 9 ° % of the fluorescence detected in the lysate is recoverable in the dialysate. Also, the experimental conditions employed in these experiments significantly increase the yield of fluorescence-producing material (i.e. Table I). It can be seen in Table VI that the H202 detected in the dialysates of phagocytizing cells is significantly greater than that detected in the dialysates of resting cells. This is true with all three particles. Biochim. Biophys. Acta, 156 (1968) 168-178
175
ESTIMATION OF H 2 0 2 IN PHAGOCYTES TABLE
VI
THE EFFECT OF PHAGOCYTOSIS
ON H 2 0 2 PRODUCTION
IN P O L Y M O R P H O N U C L E A R
LEUKOCYTES
Polymorphonuclear leukocytes dialysed against Ifrebs-Ringer phosphate m e d i u m while at o% at 37 ° resting and at 37 ° phagocytizing, all for 3 ° min. Polymorphonuclear leukocytes: particle ratio; E. cull 1:5o-1:125, p o l y s t y r e n e i :200, and S. albus i :200. .Particles
o°
Resting 37 °
Phagocytizing 37 °
Fold increase
12.o ± 2.44 (II) lO.53
3.76
H202, m#moles/Ioo I~g cellular phosphorus E. coli
1.47 ~ 0.2* ( I I )
4.27 ~- 0.62 (II) 2.80**
Polystyrene
1.26 ~: 0.03
(7)
S. albus
I.OI -¢- o . i o
(5)
4.03 -¢- 1.o5 2.77 2.98 ± 0.62 1.97
(7) (5)
9.6 ± 1.4o (7) 8.34 5 .08 ± 0.42 (5) 4.07
3 .°1 2.06
* Mean and s t a n d a r d error of the m e a n given in each case. N u m b e r in parentheses indicates the n u m b e r of experiments. ** Lower n u m b e r s represent 3 ° rain fluorescence value minus o ° values.
DISCUSSION
A method has been developed that will directly measure the H , O , content of polymorphonuclear leukocytes in the absence and presence of particulate material. By utilizing this technique, it has been shown that 2-4 times as much HzO 2 is produced by phagocytizing cells when compared to non-phagocytizing cells. These findings are considered significant as it is becoming increasingly clear that the stimulated metabolic oxidative activities of phagocytizing cells are post-phagocytic events associated with intracellular activity 5. More specifically, the increased respiratory and hexose monophosphate pathway activities exhibited by phagocytizing cells appear to be intimately involved with H20 2 formationd,5,1°. The association of these stimulated oxidative events with the intracellular killing of ingested bacteria have been demonstrated 5. However, a method to directly measure and quantitate the H20 2 content of leukocytes, at rest and during phagocytosis, had not previously been described. The direct measurement of H20 z in polymorphonuclear leukocytes or polymorphonuclear leukocytes lysates was not satisfactory. The fluorescence noted was not completely sensitive to catalase. It was felt that in order to show that fluorescence production was due to H20 2, added catalase should destroy it. This, however, could not be shown (Table I). Apparently activity of catalase is limited by component(s) present in polymorphonuclear leukocytes or the fluorescence noted was not due to H20 2. Dialysis of polymorphonuclear leukocytes and/or polymorphonuclear leukocyte lysates produced a dialysate that showed fluorescence that could be destroyed with catalase. Apparently a compound(s) is present in polymorphonuclear leukocytes that is capable of limiting catalase activity in polymorphonuclear leukocytes and further, it is non-dialyzable. Approx. 9 ° % of the total fluorescence in polymorphonuclear Biochim. Biophys. Acta, 156 (1968) 168-178
176
B. PAUL, A. J. SBARRA
leukocyte lysates or supernatant is recoverable in the dialysates and this essentially represents the total measurable H202 in the polymorphonuclear leukocytes (Table V). The experimental conditions employed in these experiments permitted the detection of increased amounts of H202 when compared to amounts detected under the initial experimental conditions (Table I). It must be emphasized that the total H202 given represents only the measurable product. The presence of catalase, myeloperoxidase and component(s) capable of limiting catalase activity in the polymorphonuclear leukocytes would suggest that the measurable H202 levels are minimum values, the actual values are probably much higher. This thesis will be developed further below. It should be noted that considerably more fluorescence producing material is recovered from lysed cells as compared to intact ceils (Tables II and III). The fact that the fluorescence-producing compound can be destroyed by catalase strongly indicates that the diffusable material is H20 ~. Additional evidence in this regard is the fact that added H20 2 can be quantitatively recovered and also destroyed by catalase. The presence of a compound limiting catalase activity in the polymorphonuclear leukocytes is most interesting. Its role may be to control the H20 2 content of the cell. Experiments designed to explore further the nature of the catalase-limiting component(s) and its role in the polymorphonuclear leukocytes are in progress. Preliminary data indicate that in the presence of an inhibitor of catalase, 3-amino1,2,4-triazole, the H202 levels are significantly higher than in the corresponding controls. This is true for both resting and phagocytizing cells. Since the dialysis procedure appeared satisfactory for the estimation of H~O2 in polymorphonuclear leukocytes, optimal conditions for its measurement needed to be established. Added H20 2 could be recovered quantitatively after a 6o-min dialysis period. In fact over 7 ° % of the added H20 2 is recovered in the dialysates after a 2o-min period of dialysis. With polymorphonuclear leukocytes, a dialysis time of 9 ° min was found to be required for maximal recovery of H,O2. No increase in fluorescence could be detected at time periods above 9 ° min (Table IV). It is apparent that maximal recovery of H20 2 is achieved only if the cell is lysed. This indicates that the endogenous and newly formed H20 e is bound in the cell and does not readily leave the cell. Possibly the newly formed H~O 2 is formed within the phagocytic vacuole where it may complex with myeloperoxidase and thereby exert its bactericidal activity. Some evidence for this thesis has been presented 1Lis. It had been previously postulated that H202 is present in polymorphonuclear leukocytes and it would appear now that this postulation is correct. Further, it had been postulated that phagocytizing polymorphonuclear leukocytes should show a higher concentration of H202 than non-phagocytizing polymorphonuclear leukocytes. This would be due to the stimulation of the hexose monophosphate pathway and other oxidative events in the phagocytizing polymorphonuclear leukocytes. Evidence that this postulation is also correct may be seen in Table VI. It should be noted that 2-4-fold increases in H20 2 production are present in phagocytizing cells. This increase cannot be attributed solely to increased release of H202 from the phagocytizing cells due to an altered permeability, as the remaining H202 in the dialysis bag, in the absence and presence of particles, was measured and was found to be essentially the same. Thus, the observed increase in H~Oe production in phagocytizing cells is primarily due to an increased formation. These results are in Biochim. Biophys. Acla, 156 (1968) 168-178
ESTIMATION OF H 2 0 2 IN PHAGOCYTES
177
accord with our recent findings that the NADP+/NADPH ratio increases significantly during phagocytosisS, 9. Since the total pyridine nucleotide level remains constant during phagocytosis, it would appear that the postulation that H , 0 2 production is the result of N A D P H oxidation is correct. Fig. 3 depicts the events leading to the production of H202 in phagocytes. GIc-6-P
I'IA I) P+
t-
'
H20t
LACTATE
Fig. 3- Postulated pathway for increased H~O 2 formation resulting from stimulated metabolic
oxidative activities.
The role of H202 as a bactericidal agent has long been known 12. The finding that there is an increased formation of this agent in phagocytizing cells is of extreme interest. In previous communications, we have presented evidence that H202 (ref. 17) and myeloperoxidase 18 are two essential agents that appear to be critical in controlling killing in the phagocyte. The present finding that HzO 2 is not only present in the phagocytes, but that it is actually increased in phagocytizing cells is compatible with this. ACKNOWLEDGEMENTS
The authors are greatly indebted to Dr. JOHNS. BAUMSTARK for constructive criticism of this manuscript. The excellent technical assistance of Mr. VIRGILIO VERZOSA is appreciated. Photography and line drawings by Mr. GEORGE DAYNES and the typing of this manuscript by Miss LINDA PARLEE are gratefully acknowledged. This investigation was supported by a grant from the Atomic Energy Commission (NY0-324o-2I) and by a U.S. Public Health Service Research Grant from the National Cancer Institute. REFERENCES H. BECKER, G. MUNDER AND H. FISCHER, Z. Physiol. Chem., 313 (1958) 266. A. J. SBARRA AND M. L. KARNOVSKY, J. Biol. Chem., 234 (1959) 1355. Z. A. COHN AND J. G. HIRSCH, J. Exptl. Med., 112 (196o) lO15. G. Y. N. IYER, M. F. ISLAM AND J. H. QUASTEL, Nature, 192 (1961) 535. R. J. SELVARAJ AND A. J. SBARRA, Nature, 211 (1966) 1272. M. ZATTI AND V. R o s s I , Experientia, 22 (1966) I. W. S. BECK, J. Biol. Chem., 232 (1958) 271. M. ZATTI AND F. RoSsI, Biochim. Biophys. Acta, 99 (1965) 577. R. J. SELVARAJ AND A. J. SBARRA, Biochim. Biophys. Acta, 141 (1967) 243. G. Y. lXT.IYER AND J. H. QUASTEL, Can. J. Biochem. physiol., 41 (1963) 427 . H. E. AEBI, Radiation Research, Suppl. 3, Argonne National Laboratory, Argonne, II1., Academic Press, New York, 1963, pp. 13o-152 . 12 G. S. WILSON ANn A. A. MILES, in TOPLEY AND WILSON, Principles of Bacteriology and Immunity, Vol. I, Williams and Wilkins, Baltimore, Md., 5th Ed. 1964, pp. 86, 87. 13 R. BRANDT AND A. S. KESTON, Anal. Biochem., I I (1965) 6. I 2 3 4 5 6 7 8 9 IO ii
Biochim. Biophys. Acta, 156 ii968) 168-178
178
B. PAUL, A. J. S BARRA
14 R. J. BEgRS, Jr. ANt) J. W. SIZER, J. Biol. Chem., 195 (1952) 133. 15 W. W. UMBREIT, R. H. BURRIS AND J. F. STAFFER, Manometric Techniques, Burgess, Minneapolis, Minn., 4 t h ed., 1964, p. 133. 16 A. S. KESTON AND R. BRANDT,Anal. Biochem., II (1965) I. 17 R. J. McRIPLEV ANt) A. J. SBARRA, J. Bacteriol., 94 (1967) 1417. 18 R. J. MCRIPLEY AND A. J. SBARRA, f. Bacteriol., 94 (1967) 1425.
Biochim. Biophys. Acta, 156 (1968) I 6 8 - 1 7 8
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25912 T H E R O L E OF T H E P H A G O C Y T E IN H O S T - P A R A S I T E I N T E R A C T I O N S X l I I . T H E D I R E C T Q U A N T I T A T I V E ESTIMATION OF H 2 0 2 IN P H A G O C Y T I Z I N G CELLS B. P A U L AND A. J. S B A R R A
Department of Pathology and Medical Research, St. Margaret's Hospital and Department of Obstetrics and Gynecology, Tufts University School of Medicine, Boston, Mass. (U.S.A .) (Received July 3ISt, 1967) (Revised manuscript received October 23rd, 1967)
SUMMARY
I. A technique that allows the direct quantitative estimation of H=O 2 in polymorphonuclear leukocytes has been developed. 2. Lysis or homogenization followed by dialysis of polymorphonuclear leukocytes results in a dialysate containing fluorescence-producing material. 3- This material is catalase sensitive. 4- Catalase specificity cannot be demonstrated without preliminary dialysis, indicating the presence of a non-dialyzable catalase-limiting component(s) in the polymorphonuclear leukocytes. 5- Phagocytizing cells contain 2-4 times as much measurable H202 as nonphagocytizing polymorphonuclear leukocytes.
INTRODUCTION
Stimulated metabolic events associated with phagocytizing cells have been well established 1-5. Increased glycolysis, oxygen consumption, oxidation of glucose through the hexose monophosphate pathway and increased formate oxidation, are major metabolic events associated with phagocytosis. In an attempt to delineate the mechanism(s) responsible for the stimulated oxidative activities, a postulation that Hz02 is a functional end-product of this oxidative metabolism was entertained 4. The H 2 0 2 would arise from the oxidation of NADPH, produced from the hexose monophosphatc pathway, b y molecular oxygen. ZATTI AND ROSSI6 have provided convincing evidence that a granule-bound NADPH-oxidase is critical in stimulating the cyanide-insensitive oxygen uptake noted during phagocytosis, and that during the process N A D P H is oxidized. BECK7 reported that NADP + is a rate-limiting factor for the hexose monophosphate pathway in human leukocytes and it has been noted in our laboratory that the addition of NADP + to guinea pig leukocyte homogenates stimulated the hexose monophosphate pathway 5. An increase in N A D P + / N A D P H ratios in phagocytizing cells has been reported by ZATTI AND ROSSI8 and by our laboratory 9. IYER AND QUASTEL 1° have Biochim. Biophys. Acta, 156 (1968) 168 178
ESTIMATION OF
H~02 IN PHAGOCYTES
169
demonstrated an enzyme system in guinea pig leukocyte homogenates capable of oxidizing N A D P H and to a lesser extent NADH, b y molecular oxygen. The increased formate oxidation exhibited b y phagocytizing cells led IYER, ISLAM AND QUASTEL 4 to predict increased H20 2 formation in these cells. It is known that formate oxidation is enhanced in the presence of H~O 2 and catalase 11. All of the above data provide indirect evidence that phagocytosis is accompanied b y an increase in H,O 2 production. The bactericidal nature of H20 2 has long been known TM. However, to the best of our knowledge a direct quantitative measure of the H,O 2 concentration in phagocytes has not been reported, Our recent finding that polymorphonuclear leukocytes are more efficient in killing Escherichia coli aerobically (supposedly when H20 2 formation is optimal) than anaerobically 5, suggested a study designed to measure directly the H20 2 concentration in resting and phagocytizing cells. This report will present the results obtained from such a study. MATERIAL AND METHODS
Reagents. All chemicals used were of reagent grade or better. Peroxidase (Horseradish) Type I I was purchased from the Sigma Chemical Co., St. Louis, Mo. and contained 127.o Purpurogallin units per mg of protein. Purified rat liver catalase was a gift from Dr. E. Kuchinskas of the State University of New York, Brooklyn, N.Y. Merck hydrogen peroxide (30 %) reagent grade was used throughout. The fluorescence-producing compound leucodiacetyl-2,7-dichlorofluorescein was synthesized according to the method of BRANDT AND KESTON13. Preparation of reagents. (I) H20 2 solutions were prepared daily by diluting 30 % H20 2 2o00 × with distilled water. The 230 m/~ absorbance of this solution was recorded. The concentration was calculated from the extinction coefficient of 81 cm -1 mole -1 at 230 m/~ and further dilutions were made in distilled H20 (ref. 14). (2) Catalase solutions were prepared in 0.025 M phosphate buffer 15 at p H 7.0. The enzyme assayed in our laboratory had the following specifications. Activity, 43 IOO units]mg protein. Protein content, 13.o mg/ml based on 407 m~ A and 430000 molar extinction coefficient. The 407/280 A ratio was o.91. In all cases I unit of catalase equals I/~mole of H20 2 decomposed/min at p H 7.0 and 25 °, assayed as described by B E E R S AND SIZER 14.
(3) Peroxidase solutions were made to i mg/ml in 0.06 M phosphate buffer at p H 6.0. (4) A stock solution of leucodiacetyl-2,7-dichlorofluorescein was made to lO-4 M in ethanol and stored at 4 °. Fluorometric determination of H202. The method of KESTON AND BRANDT16 with modifications was employed for the fluorometric assay of H,Oz in the m/, molar range. The reaction mixture, in a total of 5.0 ml contained in m~moles; phosphate buffer, I.O. lO 5 (pH 6.0) ; ZnSO 4. 7 H202, 230; leucodiacetyl-2,7-dichlorofluorescein, 8.0; H20 2 or samples from polymorphonuclear leukocytes containing HzO ~ in the range of o.o4-1.6; and peroxidase, 38 Purpurogallin units. Peroxidase was added last to start the reaction, in the absence of added horseradish peroxidase the reaction would not start, and after 2o min of incubation at room temperature, readings were recorded. Fluorometrie assays were carried out in a Beckman DU spectrophotometer with a fluorescence attachment No. 73500. For hydrogen peroxide absorbance and Biochim. Biophys. Acta, 156 (I968) I 6 8 - 1 7 8
17o
B. PAUL, A. J. SBARRA
catalase assay, a Beckman model DB speetrophotometer was used. Hydrogen peroxide and catalase solutions gave the same absorbances in both instruments. Preparation ofpolymorphonuclear neutrophils. Guinea pigs of both sexes weighing 300-400 g were used throughout. Peritoneal exudates were prepared by a previously published procedure e. The exudate containing over 9 ° % polymorphonuclear leukocytes was washed 3 times in Ca-free Krebs-Ringer phosphate medium 4 and centrifuged at 400 × g for 4 rain. The pellet was brought up to a desired volume and the absorbance at 600 mr* was determined in a Spectronic 20, Bausch and Lomb colorimeter. An A of I.O was found to be equivalent to 172/zg cellulai phosphorus (IOO/zg phosphorus is equal to 5.5" lO7 polymorphonuclear leukocytes). Test particles. IS-h cultures of E. coli and Staphylococcus albus grown in trypticase soy broth served as test particles. Before use they were autoclaved for 30 min at 121 °. The resulting heat-killed bacteria were washed twice and suspended in Krebs-Ringer phosphate medium. Polystyrene latex spherules, o . 8 I / , in diameter were purchased from the Difco Laboratories, Detroit, Mich. Lysis and dialysis of polymorphonuclear leukocytes. 4 vol. of distilled H20 (pH 5.o), were added to a polymorphonuclear leukocyte pellet (containing 50 /zg cellular phosphorus per ml) held in an ice-bath. After io rain the lysed cells were centrifuged at 12o00 × g for 20 rain. An aliquot of the supernatant was assayed for H202 directly, another aliquot was dialyzed in an ice-bath for 30 min against H20 (pH 5.o), and the dialysate assayed. Similarly, polymorphonuclear leukocytes held in Krebs-Ringer phosphate medium and dialyzed against 15-4o vol. of KrebsRinger phosphate medium were treated and examined as above. During dialysis, the polymorphonuclear leukocyte preparations were continuously agitated by means of rotating circular shaker (115 rev./min).
Estimation of total HeO 2 in polymorphonuclear leukocytes and polymorphonuclear leukocyte homogenates. Since the dialysis of polymorphonuclear leukocytes yielded dialysates suitable for H202 estimation, it was felt that studies concerned with recovery of added H202 after dialysis would be required; 12. 4 and 12.7 m/zmoles of HeO 2 in I.O ml of Krebs-Ringer phosphate medium and/ol H20 (pH 5.o), were dialyzed against 15 ml of Krebs-Ringer phosphate medium or H20 (pH 5.0) for various periods of time. For controls the same H202 concentrations were added to 15 ml of Krebs-Ringer phosphate medium. The dialysates and controls (not dialyzed) were assayed fluorimetrically. Having established optimal conditions for H 2 0 2 recovery, polymorphonuclear leukocytes samples were now dialyzed in Krebs-Ringer phosphate medium and in H20 (pH 5.o), for different periods of time and the dialysates assayed for H20 2. All dialyses were carried out at o °. Polymorphonuclear leukocytes in Krebs-Ringer phosphate medium were homogenized for 5 min using a motor-driven pestle with a teflon tip (disruption of cells was confirmed by light microscopy). An aliquot of the homogenate was dialyzed in Krebs-Ringer phosphate medium and also in distilled H~O (pH 5.o), for different periods of time; also an aliquot was added directly to Krebs-Ringer phosphate medium and to H20 (pH 5.o), and after 30 min each was centrifuged at 17300 × g for 30 min. Intact polymorphonuclear leukocytes were tested as above. All dialysates and supernatants (lysates) were assayed for H202.
Estimation of H202 production by polymorphonuclear leukocytes in the absence Biochim. Biophys. Acta, 156 (1968) 168-178
ESTIMATION OF H20 2 IN PHAGOCYTES
171
and presence of particles. Experiments designed to detect the total H , O , content of polymorphonuclear leukocytes at rest and during phagocytosis were attempted. Polymorphonuclear leukocytes in the absence and presence of particles were incubated in polyethylene tubes and after different periods of time, supernatants were assayed for H20 2. The results obtained indicated an increased H20 2 content in phagocytizing cells, however, they were not consistent. This was probably due to the complex nature of the phagocytic system (i.e. catalase, myeloperoxidase, and interfering substances present in the polymorphonuclear leukocyte preparation). In order to circumvent this problem, phagocytosis was allowed to occur in 3 different dialysis bags. One was held at o ° and served to establish the endogenous H20 2 content. The second and third bags contained polymorphonuclear leukocytes with and without different particles and they were both incubated at 37 ° . Heatkilled E. coli, S. albus and polystyrene latex spherules served as test particles. The resulting dialysates were assayed for H20 2. RESULTS
The fluorescence obtained when known concentrations of H,O 2 were assayed are plotted in Fig. I. A linear relationship exists at low HzO 2 concentrations and this part of the curve was always used for the analyses.
0 0
20
40
60
~ , mpmoies x 1110
80
i00
Fig. I. Relationship between fluorescence and hydrogen peroxide concentrations. 30% H202 (Merck) diluted with distilled H~O. Fluorescence measured after 20 min incubation at 25 °. Fluorescence plotted against re#moles of H202 contained in 5 ml of the assay mixture.
Direct measurement of H~O 2 in polymorphonuclear leukocytes lysates was found to be satisfactory, but the observed fluorescence was not destroyed b y catalase and the addition of H202 to the lysate did not increase fluorescence. It would appear that interfering substances are present in polymorphonuclearleukocyte lysates. These results m a y be seen in Table I. In an attempt to circumvent the problem of interfering material in polymorphonuclear leukocytes, dialysis of polymorphonuclear leukocytes under different experimental conditions was tried. Fluorescence was noted in the dialysate and further Biochim. Biophys. dora, 156 (i968) 168-178
I72
B. PAUL, A. J. SBARRA
TABLE I FLUORESCENCE
PRODUCED
BY POLYMORPHONUCLEAR
Sample
H202, re#moles*
Lysate * * Lysate -I- catalase § Lysate + HlO~ §§ Lysate + H~O 2 + catalase
6.59 7.38 6.38 6.95
LEUKOCYTE
LYSATE
~ 0.64*** ± 0.60 ± 0.59 4:0.45
All results adjusted for IOO/zg cellular phosphorus or 5.5" lO7 cells. ** Io min after addition of 4 vol. of H20, pH 5.o at o °, the lysed cells were centrifuged at i2 ooo × g for 2o min and the supernatant (lysate) assayed. Readings were taken after a 2o min reaction time. Lysate from I-iO/2g cellular phosphorus per assay mixture. *** Mean ~- standard error of the mean of 3 experiments are presented in each case. § 0.75 unit of rat liver catalase per assay mixture. 15 nfin after addition of catalase (at 25 °) samples were assayed. §§ o.I6 or 0.32 m/~mole of H~O 2 (Merck) per assay mixture. See text for additional details. T A B L E II FLUORESCENCE PRODUCED BY POLYMORPHONUCLEAR LEUKOCYTE DIALYSATE
Treatment
H202, m~moles*
A. Polymorphonuclear leukocytes dialysed in Krebs-Ringer phosphate medium for 3 ° min. Dialysate assayed
1.56 ± o. I3 (I4)**
B. Polymorphonuclear leukocytes lysed with H20, pH 5.0. Supernatant of the lysate***: i. Dialysed in H20 (pH 5.o) for 3 ° min and dialysate assayed 2. Assayed directly
4.84 ~: 0.62 7.46 =t= 0.22
(6) (9)
* Dialysate or lysate from i - i o / ~ g cellular phosphorus assayed in each case and results adjusted for ioo/zg cellular phosphorus. ** Mean and standard error of the mean given in each case. The number in parentheses indicates the number of experiments performed. *** io min after addition of 4 vol. of H20 (pH 5.0) the lysed cells were centrifuged at 12 ooo × g for 20 rain and the supernatant was treated as indicated. Readings taken after 20 min reaction time. See Table I and text for additional details. i t w a s n o t e d t h a t l y s e d cells a f t e r d i a l y s i s s h o w e d g r e a t e r f l u o r e s c e n c e t h a n n o n - l y s e d cells. T h e s e r e s u l t s a r e s h o w n i n T a b l e I I . I n o r d e r t o a s c e r t a i n if t h e f l u o r e s c e n c e n o t e d i n t h e d i a l y s a t e s w a s d u e t o H20~ and not to a non-specific material, an attempt to destroy the fluorescent-producing material with catalase was studied. It can be seen in Table III that the catalasetreated dialysates showed essentially no fluorescence. This would appear to indicate t h a t t h e f l u o r e s c e n c e n o t e d i n t h e d i a l y s a t e s w a s d u e t o H 2 0 ~. T h e a d d i t i o n of 0.75 u n i t of t h e r a t l i v e r c a t a l a s e p e r a s s a y m i x t u r e w a s f o u n d n o t t o i n t e r f e r e w i t h t h e a s s a y s y s t e m w h i l e a t t h e s a m e t i m e i t w a s c a p a b l e of d e s t r o y i n g t h e f l u o r e s c e n c e p r o d u c i n g m a t e r i a l . S a m p l e s w e r e a s s a y e d a t 25 o, 15 m i n a f t e r t h e a d d i t i o n of c a t a l a s e . T o e s t a b l i s h f u r t h e r o p t i m a l c o n d i t i o n s for a s s a y , H 2 0 , r e c o v e r y e x p e r i m e n t s w e r e c a r r i e d o u t . I t c a n b e s e e n i n Fig. 2 t h a t i n 60 r a i n 90 % of t h e H 2 0 2 a d d e d t o a d i a l y s i s b a g is r e c o v e r e d i n t h e d i a l y s a t e . T h e effect of t h e t i m e of d i a l y s i s o n H 2 0 2
Biochim. Biophys. Acta, 156 (1968) i 6 8 - i 7 8
173
ESTIMATION OF H 2 0 2 IN PHAGOCYTES TABLE III DESTRUCTION LEUKOCYTE
OF
THE
DIALYSATES
FLUORESCENCE-PRODUCING BY
COMPONENT
(H202) OF
POLYMORPHONUCLEAR
CATALASE
Samples
HzO~, re#moles*
A. I. 2. 3. 4.
Krebs-Ringer Krebs-Ringer Krebs-Ringer Krebs-Ringer
B. I. 2. 3. 4.
Dialysate Dialysate Dialysate Dialysate
phosphate phosphate phosphate phosphate
medium medium medium medium
dialysates 1.63 ± o.3** (8) 0.02 (8) d i a l y s a t e s + c a t a l a s e * ** dialysates + HzO 2§ 97 % H202 recovered (3) d i a l y s a t e s + H202 + catalase o.o (3)
of l y s a t e of l y s a t e + catalase of l y s a t e + H202 of l y s a t e + H202 -[- catalase
3.8 ± 0.06 (8) 0.20 (3) 82 % H 2 0 ~ recovered (3) 5-7% H2Oi recovered (3)
(A) p o l y m o r p h o n u c l e a r l e u k o c y t e s d i a l y s e d in K r e b s - R i n g e r p h o s p h a t e m e d i u m for 3 ° m i n a t o °. D i a l y s a t e s a n a l y s e d as indicated. (B) p o l y m o r p h o n u c l e a r l e u k o c y t e s l y s e d w i t h H 2 0 (pH 5.0) (as in T a b l e I**). S u p e r n a t a n t dialysed for 30 rain a t o ° a n d a n a l y s e d as indicated. * R e s u l t s a d j u s t e d for ioo # g cellular p h o s p h o r u s . ** M e a n a n d s t a n d a r d error of t h e m e a n g i v e n in each case. N u m b e r in p a r e n t h e s e s refers to t h e n u m b e r of e x p e r i m e n t s . *** 0.75 u n i t of t h e r a t liver catalase per a s s a y m i x t u r e . 15 m i n after a d d i t i o n of c a t a l a s e (at 25 °) s a m p l e s were assayed. § o.16 a n d 0.32 m # m o l e of H 2 0 ~ (Merck) per a s s a y m i x t u r e . See Table I a n d t e x t for a d d i t i o n a l details. 100
IlO
.
~
60
M
100
DIALYSIS TIME, MINUTES
Fig. 2. R e c o v e r y of a d d e d h y d r o g e n peroxide (Merck) f r o m d i a l y s a t e s . 12. 4 a n d 12. 7 m/zmoles of H202 c o n t a i n e d in I.O m l of K r e b s - R i n g e r p h o s p h a t e m e d i u m , dialyzed a g a i n s t 15.o m l of K r e b s R i n g e r p h o s p h a t e m e d i u m . F o r controls t h e s a m e c o n c e n t r a t i o n s of H~O 2 directly a d d e d to 15.0 ml of K r e b s - R i n g e r p h o s p h a t e m e d i u m . T h e d i a l y s a t e s a n d controls a s s a y e d fluorometrically a n d r e s u l t s are p r e s e n t e d as p e r c e n t of control.
release by polymorphonuclear leukocytes after dialysis in Krebs-Ringer phosphate medium and in H20 (pH 5.0) is shown in Table IV. It is evident that lysed cells, (H20, pH 5.0) release more H20 2 than cells held in Krebs-Ringer phosphate medium, where the cells remain intact. With lysed ceils maximal release of H~O2 (dialysis) was achieved in 9° min. This time period was used in all subsequent experiments. Biochim. Biophys. Acta, 156 (1968) 168-178
174
B. PAUL, A. J. SBARRA
T A B L E IV THE
]~FFECT OF TIME
Dialysis time (min)
IO 20 3° 45 60 90
OF DIALYSIS
ON
H202
RELEASE
BY POLYMORPHONUCLEAR
LEUKOCYTES
H202, re#moles* dialysed in Krebs-Ringer phosphate medium 1.9 2. 3 2. 7 2. 9 3.6 3.9
(5)** (8) (8) (8) (8) (5)
H20 (pH 5.0)
3 .2 (4) 4 .0 (5) 5.4 (5) 9.5 (II) 11. 4 (IO)
* R e s u l t s a d j u s t e d for IOO/~g cellular p h o s p h o r u s . ** Mean a n d n u m b e r of e x p e r i m e n t s (in p a r e n t h e s e s ) g i v e n in each case. See Table I a n d t e x t for a d d i t i o n a l details.
TABLE V TOTAL MEASURABLE H202 LEUKOCYTE HOMOGENATES
OF
POLYMORPHONUCLEAR
Treatment
LEUKOCYTES
Polymorphonuclear leukocytes
AND
POLYMORPHONUCLEAR
Homogenate
H202 ml~moles/Ioo ttg cellular phosphorus Dialysis in K r e b s - R i n g e r p h o s p h a t e m e d i u m * Dialysis in H 2 0 (pH 5.o) Direct addition to H 2 0 (pH 5.0)***
3.90 (5)** ~3.o (8) I5.54 (4)
12.4 (5) 14.4 (8) 15.6o (6)
* Dialysis in all cases were carried o u t a t o ° for 9o min. D i a l y s a t e s assayed. ** M e a n a n d n u m b e r of e x p e r i m e n t s (in parentheses) g i v e n in each case. *** Direct a d d i t i o n to 15 vol. of H~O (pI-t 5.0). 3 ° rain after a d d i t i o n a n d at o °, c e n t r i f u g e d in t h e cold at 17 300 × g for 30 min. S u p e r n a t a n t s assayed. See Table I a n d t e x t for a d d i t i o n a l details.
An attempt was made to estimate the total measurable fluorescence-producing material (H202) of polymorphonuclear leukocytes and polymorphonuclear leukocyte homogenates. These results are presented in Table V. It appears that no significant differences are evident between the different experimental conditions. It should be noted that approx. 9 ° % of the fluorescence detected in the lysate is recoverable in the dialysate. Also, the experimental conditions employed in these experiments significantly increase the yield of fluorescence-producing material (i.e. Table I). It can be seen in Table VI that the H202 detected in the dialysates of phagocytizing cells is significantly greater than that detected in the dialysates of resting cells. This is true with all three particles. Biochim. Biophys. Acta, 156 (1968) 168-178
175
ESTIMATION OF H 2 0 2 IN PHAGOCYTES TABLE
VI
THE EFFECT OF PHAGOCYTOSIS
ON H 2 0 2 PRODUCTION
IN P O L Y M O R P H O N U C L E A R
LEUKOCYTES
Polymorphonuclear leukocytes dialysed against Ifrebs-Ringer phosphate m e d i u m while at o% at 37 ° resting and at 37 ° phagocytizing, all for 3 ° min. Polymorphonuclear leukocytes: particle ratio; E. cull 1:5o-1:125, p o l y s t y r e n e i :200, and S. albus i :200. .Particles
o°
Resting 37 °
Phagocytizing 37 °
Fold increase
12.o ± 2.44 (II) lO.53
3.76
H202, m#moles/Ioo I~g cellular phosphorus E. coli
1.47 ~ 0.2* ( I I )
4.27 ~- 0.62 (II) 2.80**
Polystyrene
1.26 ~: 0.03
(7)
S. albus
I.OI -¢- o . i o
(5)
4.03 -¢- 1.o5 2.77 2.98 ± 0.62 1.97
(7) (5)
9.6 ± 1.4o (7) 8.34 5 .08 ± 0.42 (5) 4.07
3 .°1 2.06
* Mean and s t a n d a r d error of the m e a n given in each case. N u m b e r in parentheses indicates the n u m b e r of experiments. ** Lower n u m b e r s represent 3 ° rain fluorescence value minus o ° values.
DISCUSSION
A method has been developed that will directly measure the H , O , content of polymorphonuclear leukocytes in the absence and presence of particulate material. By utilizing this technique, it has been shown that 2-4 times as much HzO 2 is produced by phagocytizing cells when compared to non-phagocytizing cells. These findings are considered significant as it is becoming increasingly clear that the stimulated metabolic oxidative activities of phagocytizing cells are post-phagocytic events associated with intracellular activity 5. More specifically, the increased respiratory and hexose monophosphate pathway activities exhibited by phagocytizing cells appear to be intimately involved with H20 2 formationd,5,1°. The association of these stimulated oxidative events with the intracellular killing of ingested bacteria have been demonstrated 5. However, a method to directly measure and quantitate the H20 2 content of leukocytes, at rest and during phagocytosis, had not previously been described. The direct measurement of H20 z in polymorphonuclear leukocytes or polymorphonuclear leukocytes lysates was not satisfactory. The fluorescence noted was not completely sensitive to catalase. It was felt that in order to show that fluorescence production was due to H20 2, added catalase should destroy it. This, however, could not be shown (Table I). Apparently activity of catalase is limited by component(s) present in polymorphonuclear leukocytes or the fluorescence noted was not due to H20 2. Dialysis of polymorphonuclear leukocytes and/or polymorphonuclear leukocyte lysates produced a dialysate that showed fluorescence that could be destroyed with catalase. Apparently a compound(s) is present in polymorphonuclear leukocytes that is capable of limiting catalase activity in polymorphonuclear leukocytes and further, it is non-dialyzable. Approx. 9 ° % of the total fluorescence in polymorphonuclear Biochim. Biophys. Acta, 156 (1968) 168-178
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B. PAUL, A. J. SBARRA
leukocyte lysates or supernatant is recoverable in the dialysates and this essentially represents the total measurable H202 in the polymorphonuclear leukocytes (Table V). The experimental conditions employed in these experiments permitted the detection of increased amounts of H202 when compared to amounts detected under the initial experimental conditions (Table I). It must be emphasized that the total H202 given represents only the measurable product. The presence of catalase, myeloperoxidase and component(s) capable of limiting catalase activity in the polymorphonuclear leukocytes would suggest that the measurable H202 levels are minimum values, the actual values are probably much higher. This thesis will be developed further below. It should be noted that considerably more fluorescence producing material is recovered from lysed cells as compared to intact ceils (Tables II and III). The fact that the fluorescence-producing compound can be destroyed by catalase strongly indicates that the diffusable material is H20 ~. Additional evidence in this regard is the fact that added H20 2 can be quantitatively recovered and also destroyed by catalase. The presence of a compound limiting catalase activity in the polymorphonuclear leukocytes is most interesting. Its role may be to control the H20 2 content of the cell. Experiments designed to explore further the nature of the catalase-limiting component(s) and its role in the polymorphonuclear leukocytes are in progress. Preliminary data indicate that in the presence of an inhibitor of catalase, 3-amino1,2,4-triazole, the H202 levels are significantly higher than in the corresponding controls. This is true for both resting and phagocytizing cells. Since the dialysis procedure appeared satisfactory for the estimation of H~O2 in polymorphonuclear leukocytes, optimal conditions for its measurement needed to be established. Added H20 2 could be recovered quantitatively after a 6o-min dialysis period. In fact over 7 ° % of the added H20 2 is recovered in the dialysates after a 2o-min period of dialysis. With polymorphonuclear leukocytes, a dialysis time of 9 ° min was found to be required for maximal recovery of H,O2. No increase in fluorescence could be detected at time periods above 9 ° min (Table IV). It is apparent that maximal recovery of H20 2 is achieved only if the cell is lysed. This indicates that the endogenous and newly formed H20 e is bound in the cell and does not readily leave the cell. Possibly the newly formed H~O 2 is formed within the phagocytic vacuole where it may complex with myeloperoxidase and thereby exert its bactericidal activity. Some evidence for this thesis has been presented 1Lis. It had been previously postulated that H202 is present in polymorphonuclear leukocytes and it would appear now that this postulation is correct. Further, it had been postulated that phagocytizing polymorphonuclear leukocytes should show a higher concentration of H202 than non-phagocytizing polymorphonuclear leukocytes. This would be due to the stimulation of the hexose monophosphate pathway and other oxidative events in the phagocytizing polymorphonuclear leukocytes. Evidence that this postulation is also correct may be seen in Table VI. It should be noted that 2-4-fold increases in H20 2 production are present in phagocytizing cells. This increase cannot be attributed solely to increased release of H202 from the phagocytizing cells due to an altered permeability, as the remaining H202 in the dialysis bag, in the absence and presence of particles, was measured and was found to be essentially the same. Thus, the observed increase in H~Oe production in phagocytizing cells is primarily due to an increased formation. These results are in Biochim. Biophys. Acla, 156 (1968) 168-178
ESTIMATION OF H 2 0 2 IN PHAGOCYTES
177
accord with our recent findings that the NADP+/NADPH ratio increases significantly during phagocytosisS, 9. Since the total pyridine nucleotide level remains constant during phagocytosis, it would appear that the postulation that H , 0 2 production is the result of N A D P H oxidation is correct. Fig. 3 depicts the events leading to the production of H202 in phagocytes. GIc-6-P
I'IA I) P+
t-
'
H20t
LACTATE
Fig. 3- Postulated pathway for increased H~O 2 formation resulting from stimulated metabolic
oxidative activities.
The role of H202 as a bactericidal agent has long been known 12. The finding that there is an increased formation of this agent in phagocytizing cells is of extreme interest. In previous communications, we have presented evidence that H202 (ref. 17) and myeloperoxidase 18 are two essential agents that appear to be critical in controlling killing in the phagocyte. The present finding that HzO 2 is not only present in the phagocytes, but that it is actually increased in phagocytizing cells is compatible with this. ACKNOWLEDGEMENTS
The authors are greatly indebted to Dr. JOHNS. BAUMSTARK for constructive criticism of this manuscript. The excellent technical assistance of Mr. VIRGILIO VERZOSA is appreciated. Photography and line drawings by Mr. GEORGE DAYNES and the typing of this manuscript by Miss LINDA PARLEE are gratefully acknowledged. This investigation was supported by a grant from the Atomic Energy Commission (NY0-324o-2I) and by a U.S. Public Health Service Research Grant from the National Cancer Institute. REFERENCES H. BECKER, G. MUNDER AND H. FISCHER, Z. Physiol. Chem., 313 (1958) 266. A. J. SBARRA AND M. L. KARNOVSKY, J. Biol. Chem., 234 (1959) 1355. Z. A. COHN AND J. G. HIRSCH, J. Exptl. Med., 112 (196o) lO15. G. Y. N. IYER, M. F. ISLAM AND J. H. QUASTEL, Nature, 192 (1961) 535. R. J. SELVARAJ AND A. J. SBARRA, Nature, 211 (1966) 1272. M. ZATTI AND V. R o s s I , Experientia, 22 (1966) I. W. S. BECK, J. Biol. Chem., 232 (1958) 271. M. ZATTI AND F. RoSsI, Biochim. Biophys. Acta, 99 (1965) 577. R. J. SELVARAJ AND A. J. SBARRA, Biochim. Biophys. Acta, 141 (1967) 243. G. Y. lXT.IYER AND J. H. QUASTEL, Can. J. Biochem. physiol., 41 (1963) 427 . H. E. AEBI, Radiation Research, Suppl. 3, Argonne National Laboratory, Argonne, II1., Academic Press, New York, 1963, pp. 13o-152 . 12 G. S. WILSON ANn A. A. MILES, in TOPLEY AND WILSON, Principles of Bacteriology and Immunity, Vol. I, Williams and Wilkins, Baltimore, Md., 5th Ed. 1964, pp. 86, 87. 13 R. BRANDT AND A. S. KESTON, Anal. Biochem., I I (1965) 6. I 2 3 4 5 6 7 8 9 IO ii
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14 R. J. BEgRS, Jr. ANt) J. W. SIZER, J. Biol. Chem., 195 (1952) 133. 15 W. W. UMBREIT, R. H. BURRIS AND J. F. STAFFER, Manometric Techniques, Burgess, Minneapolis, Minn., 4 t h ed., 1964, p. 133. 16 A. S. KESTON AND R. BRANDT,Anal. Biochem., II (1965) I. 17 R. J. McRIPLEV ANt) A. J. SBARRA, J. Bacteriol., 94 (1967) 1417. 18 R. J. MCRIPLEY AND A. J. SBARRA, f. Bacteriol., 94 (1967) 1425.
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