Biochimica et Biophysica Acta, 584 (1979) 143--148 © Elsevier/North-Holland Biomedical Press
BBA 28862 ROLE OF CYTOSKELETAL ELEMENTS IN CYTOCHALASIN E-INDUCED SUPEROXIDE PRODUCTION BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES AKIRA NAKAGAWARAa,, and SHIGEKI MINAKAMIb a Department of Surgery, Division of Pediatric Surgery and b Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812 (Japan)
(Received August 28th, 1978) Key words; Cytochalasin E; Superoxide ion; Microtubule; Microfilament; Phagocytosis; (Leukocyte; Cytoskeletal element)
Summary The release of superoxide anions from human polymorphonuclear leukocytes induced by cytochalasin E was greatly enhanced by the pretreatment of the cells either with deuterium oxide or with concanavalin A. Colchicine, vinblastine and cyclic AMP inhibited the release. Cytochalasins A and B also suppressed the superoxide release. These observations suggest the involvement of microfilament-microtubule system in the production and release of superoxide anions induced by cytochalasin E.
Introduction Recent investigations have revealed that microtubules and micro filaments control some intracellular events by influencing membrane fluidity . Hoffstein et al.  have shown that concanavalin A which binds to cell surface glycoproteins, induces the assembly of microtubules and the discharge of specific granules in polymorphonuclear leukocytes. Capping of the cells induced by concanavalin A is also affected by the treatment of the cells with colchicine which destroys microtubule assembly . In previous publications [4,5], we have reported that the production and release of superoxide anions (O~) are induced by the treatment of polymorphonuclear leukocytes with cytochalasin E, while the ingestion of particles is completely inhibited by the antibiotic. The 02 release has apparently similar char* T o w h o m reprint requests s h o u l d be a d d r e s s e d .
144 acteristics as phagocytic metabolism, accompanying an increase in cyanideinsensitive respiration and a stimulation of hexose monophosphate oxidative pathway. This report presents observations which suggest that the production and release of O~ from human polymorphonuclear leukocytes induced by cytochalasin E is modulated by cytoskeletal elements: microtubules and microfilaments. Materials and Methods
Materials. Superoxide dismutase (EC 126.96.36.199) was prepared from ox blood by the method of McCord and Fridovich . Cytochalasins A, B and E were obtained from Aldrich Chemical Co., concanavalin A and vinblastine sulfate from Sigma Chemical Co., colchicine and deuterium oxide (2H20) from Merck Chemical Co., and cytochrome c and cyclic adenine nucleotides from Boehringer und SShne. Other reagents were of analytical reagent grade. Preparation of leukocytes. Human leukocytes were obtained from normal subjects by means of dextran sedimentation and hypotonic lysis . Venous blood (10 ml) was added to the same volume of 3% dextran-saline solution and kept at room temperature for 30 min. The supernatant was centrifuged at 170 × g for 7 min and the pellet was suspended in a small volume of saline solution buffered with 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4 (HEPES-saline). After contaminated red cells were removed by hypotonic lysis, leukocytes were washed with 10 ml of HEPES-saline solution. The leukocytes were finally suspended in a small volume of ice-cold HEPES-saline solution. Assay of released superoxide anions. The superoxide anions released from leukocytes were measured by the reduction of exogenous ferricytochrome c at 550--540 nm on a dual wavelength spectrophotometer (Hitachi 556) with a constant temperature cuvette holder kept at 37°C. A molar absorption coefficient of cytochrome c was taken as 19.1 • 103. Experimental conditions are described as figure legends. Results
Stimulation of the O~ release by treatment with 2H20 and concanavalin A Superoxide anions were released from human polymorphonuclear leukocytes when the cells were treated with cytochalasin E, as reported previously . The reduction of exogenous ferricytochrome c was observed after the addition of cytochalasin E in three phases: (1) an initial transient release of O~, (2) a lag phase which continues several minutes, and (3) a steady-state release which gives essentially a linear increase of the reduced pigment. The reduction of cytochrome c was completely inhibited by the addition of superoxide dismutase (Fig. 1A). When we tried a similar experiment in HEPES-saline solution containing 70% 2H20, the lag phase disappeared and the steady-state rate of the O~ release was markedly enhanced (Fig. 1B). Superoxide dismutase not only inhibited the reduction of ferricytochrome c, but also caused the oxidation of the reduced
/ Cyt. E
D20 (70"/,) Fig. 1. The time-courses of the cytochalasin-induced O~ release from h u m a n polymorphonucleax leukocytes treated with 2 H 2 0 ( D 2 0 ) and concanavalin A (Con. A). The cells ( 1 - 1 0 6 ) were suspended in 1.0 ml HEPES-saline solution containing 2 mM glucose, 65 pM ferricytochrome c and 1 mM CaC12 . Cytochaiasin E solution (2 mg/ml of dimethylsulfoxide) was diluted with the same volume of HEPES-saline solution immediately before use and 5 #1 was added at the points indicated by the arrows labelled as Cyt. E. Dotted lines indicate the experiments with the same concentration of dimethylsulfoxide. Supezoxide dismutase (10 #g) was added at the points indicated by the arrows labelled as SOD. (A) Control experiment. (B) Experiment in the presence of 70% 2H20. (C) Experiment with concanavalin A (50 pg) which was added 3 min before the addition of cytochaiasin E, at the point indicated by the arrow labelled as Con. A.
pigment. This may be ascribed to the accumulation of H202 at high concentrations, because the rate of the O~ release is so fast and O~ is converted to H202 by superoxide dismutase. The half maximal stimulation was observed at about 50% 2H20 and the maximal effect at about 65% 2H20. The stimulation up to 50-fold was observed, when the rate of the cytochrome c reduction at 4 min after the addition of cytochalasin E was compared (Fig. 2). Although 2H20
, 40 D20
Fig. 2. Stimulation of the O~ release by different concentrations of 2 H 2 0 (D20). Experimental conditions were essentially the same as in Fig. 1, except the numbers of the cells (1 • 105) and the concentrations of 2 H 2 0 which are given on the abscissa. The rate of cytochrome c reduction at 4 rain after the addition of cytochalasin E is given as the rate of the O~ release (the ordinate).
stimulated the release in the absence of cytochalasin E, the stimulation was not so strong as observed in guinea pig leukocytes of peritoneal exudates . Similarly, the pretreatment of the cells with concanavalin A (50 pg/ml) caused the disappearance of the lag phase and stimulated greatly the steadystate rate of the O~ release induced by the cytochalasin (Fig. 1C). Fig. 3 shows the effect of concanavalin A concentration on the rate of O~ release at 5 min after the addition of the cytochalasin. The curve is apparently sigmoidal and the concentration of the lectin which gives half maximal stimulation was about 40 tzg/ml. The activation by the lectin was observed just after the addition and the effect was maximal at about 30 s. It gradually decreased thereafter and became constant after about 7 min.
Inhibition o f the O~ release by colchicine and vinblastine Colchicine or vinblastine inhibited the O~ release induced by cytochalasin E, when leukocytes were pretreated with the reagent for 15 min at 37°C. The initial transient release of O~ after the addition of the cytochalasin disappeared by the pretreatment, while the steady-state rate of the O~ release was only moderarely affected: about 30% inhibition of the steady-state rate was observed with 100 pM colchicine or 5 pM vinblastine. Effect o f cyclic AMP on the O~ release The O~ release induced by cytochalasin E was inhibited by 3',5'-cyclic AMP. The rate of cytochrome c reduction was inhibited by 0.2 mM theophylline to about 80% of the control and the addition of cyclic AMP or dibutyryl cyclic AMP synergistically inhibited the reduction rate (Fig. 4). The treatment of leukocytes with papaverine, another inhibitor of phosphodiesterase, also suppressed the reduction of cytochrome c: the inhibition of the steady-state rate
2.0 c E -5 E
0.5 / ~ o / / ~ 0
t t 4.0 60 Concanavatin A
i , t 80 100 ( I,Jg/mt )
Fig. 3. S t i m u l a t i o n o f t h e O~ r e l e a s e b y t h e p r e t r e a t m e n t o f t h e cells w i t h c o n c a n a v a l i n A of d i f f e r e n t c o n c e n t r a t i o n s . E x p e r i m e n t a l c o n d i t i o n s w e r e e s s e n t i a l l y t h e s a m e as i n Fig. 1, e x c e p t t h e n u m b e r s o f t h e cells (2 • 10 5) a n d t h e c o n c e n t r a t i o n s o f c o n c a n a v a l l n A w h i c h are g i v e n o n t h e a b s c i s s a . C y t o c h a l a s i n E (o e, 5/~g; o . . . . . . o, 2/~g) w a s a d d e d at 1 m i n a f t e r t h e a d d i t i o n o f c o n c a n a v a l i n A. T h e r a t e o f c y t o c h r o r n e c r e d u c t i o n at 5 r a i n a f t e r t h e a d d i t i o n o f c y t o c h a t a s i n E is g i v e n as t h e r a t e o f t h e O~ release (the ordinate).
147 100 -6
C o n c e n t r a t i o n (rnM) Fig. 4 . I n h i b i t i o n o f t h e O 5 r e l e a s e b y c y c l i c A M P a n d d i b u t y l c y l c y c l i c A M P i n t h e p r e s e n c e o f t h e o p h y l line. E x p e r i m e n t a l c o n d i t i o n s w e r e e s s e n t i a l l y t h e s a m e as i n Fig. 1 , e x c e p t the n u m b e r s o f t h e cells (4 • 1 0 6 ) . T h e celis w e r e p r e i n c u b a t e d in t h e p r e s e n c e o f 0 . 2 m M t h e o p h y l l i n e a n d c y c l i c n u c l e o t i d e s , t h e c o n c e n t z a t i o n s o f w h i c h axe given o n t h e a b s c i s s a , f o r 8 r n i n a t 3 7 ° C . T h e r a t e o f t h e O~ r e l e a s e a t 1 2 r a i n a f t e r t h e a d d i t i o n o f e y t o c h a l a s i n E w a s g i v e n o n t h e o r d i n a t e as p e r c e n t a g e o f t h e c o n t r o l r a t e w i t h o u t cyclic nucleotides (0.75 nmol/min), o o, c y c l i c A M P ; z~ ~, d i b u t y r y l c y c l i c A M P .
was about 60% at 20 pM and more than 80% at 50 ~M of papaverine, when the cells were preincubated with the inhibitor for 10 min at 37°C.
Effect o f cytochalasins A and B on the O~ release When polymorphonuclear leukocytes Were pretreated at 37°C for 10 min with cytochalasin A or cytochalasin B, another member of cytochalasin family which affects microfilaments but does not induce the O~ release , the release induced by cytochalasin E was strongly inhibited. The transient release of O~ after the addition of cytochalasin E disappeared and the inhibition of the steady-state rate was about 50% by cytochalasin B at 2.5 pg/ml and more than 80% by cytochalasin A at 5 pg/ml. Discussion
The production of O~ by polymorphonuclear leukocytes is one of phagocytic metabolism which is involved in the intracellular killing of bacteria. Previously, we have shown that the treatment of the cells with cytoehalasin E induces the O~ release whose characteristics are similar to that of the O~ release associated with phagocytosis, though the antibiotic itself inhibits the particle ingestion [4,5]. The O~ release induced by the cytoehalasin was not observed in polymorphonuclear leukoeytes of patients with chronic granulomatous disease . The cytochalasin seems to accelerate both the production and the release of O~, but we designate the phenomena as the release, because we can only measure the appearance of O~ in the medium. The antibiotic induces the formation of zeiotie knobs and the release of lysosomal enzymes, without apparent destruction of the cells and the release of cytosol enzymes such as lactate dehydrogenase [ 5]. It has generally been accepted that some cytoplasmic phenomena are controlled by plasma membrane through cytoskeletal components, in particular the mierofilament-microtubule system [ 1]. Complicated events such as capping and aggregation seem to require transmembrane communication and structural
linkage with the cytoskeletal elements. Involvement of microtubule system in secretory process of human leukocytes has been suggested: Hoffstein et al.  have shown that concanavalin A induces microtubule assembly in the cells, accompanying a selective discharge of specific granules, and Zurier et al.  have shown that 2H20 enhances the secretion through polymerization or stabilization of microtubules and the secretion is inhibited by colchicine or vinblastine. The present report indicates that the release of O~ induced by cytochalasin E was greatly enhanced by the treatment of the cells with either concanavalin A or 2H20 and was suppressed by microtubule-disrupting agents such as colchicine or vinblastine. Synergistic inhibition of the 02 release by cyclic AMP and theophylline supports the supposition that microtubule system is involved in the production and the release of 02, because cyclic AMP is known to inhibit microtubule assembly . The microfilament system also appears to be involved in the production and release of 02, because cytochalasins which are known to exert effects on microfilaments [11,12] affected the 02 release: cytochalasin E induced the release whereas cytochalasins A and B suppressed the release. All the observations given in the present report suggest that the production and release of O~ by polymorphonuclear leukocytes is controlled by cytoskeletal components, microfilament-microtubule system, though further research is necessary to confirm the supposition.
Acknowledgements This study was supported in part by the Yamanouchi Foundation of Metabolism and Diseases and Science Research Grants from the Ministry of Education, Science and Culture of Japan.
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