J. Biochem. 86, 469-475 (1979)
Evidence for Bactericidal Activity of Polymorphonuclear
Naoki OKAMURA, Sadahiko ISHLBASHI, and Tatsuya TAKANO1 Department of Physiological Chemistry, Hiroshima University School of Medicine, Kasumi, Hiroshima, Hiroshima 734 Received for publication, February 9, 1979
The relationship between phagocytosis and bactericidal action of polymorphonuclear leukocytes was examined by comparing the functions of cytochalasin D-treated leukocytes with those of the control. Measurement of phagocytotic and bacterial DNA-degrading activities using Escherichia coli prelabeled with PHJthymidine revealed that phagocytosis and bacterial DNA degradation were inhibited by treatment with cytochalasin D to about 50 and 10% of the control, respectively. Nevertheless, the bactericidal activity of the cytochalasin D-treated leukocytes was almost the same as that of the control leukocytes; almost all the bacteria were phagocytized by the latter leukocytes. Under the same experimental conditions, the production and release of superoxide anions and hydrogen peroxide, which are both known to be involved in the bactericidal action of the leukocytes, were markedly increased by cytochalasin D. Release of several lysosomal hydrolases was also increased markedly by cytochalasin D treatment, except for myeloperoxidase. However, lactate dehydrogenase, a typical cytosolic marker, was not released by the same treatment. Thus, it is unlikely that the increase in the release of the above-mentioned bactericidal factors was due to decomposition of the leukocytes. These results indicate that the site of bactericidal action of cytochalasin D-treated leukocytes is not necessarily intracellular but may be around the external surface of the cells.
In connection with the bactericidal function of polymorphonuclear leukocytes (PMNL), there is considerable interest in the involvement of reactive oxygen metabolites, such as superoxide anion, hydrogen peroxide, singlet oxygen, hydroxyl radical, etc., which are believed to be formed accompanying phagocytosis (1-8). The roles of lysosomal enzymes, such as lysozyme, cathepsins, etc., and of cationic proteins have also been con•Present address: Faculty of Pharmaceutical Sciences, Teikyo University, Tsukui, Kanagawa 199-01. Abbreviations: PMNL, polymorphonuclear leukocyte, Vol. 86, No. 2, 1979
469
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Leukocytes without Phagocytosis
sidered in the bactericidal action of PMNL (9, 10). On the other hand, it was reported that cytochalasin E as well as cytochalasin D stimulated superoxide anion production in PMNL, though both inhibited phagocytosis (11, 12). In fact, it has not yet been fully clarified how and where phagocytosis and bacteria-killing by these factors proceed in PMNL, though it is generally believed that bacteria are killed after phagocytosis and are successively digested in phagolysosomes. In this paper, we present evidence that bacteria may be killed by PMNL without phagocytosis, on the basis of findings that cytochalasin D-treated
470
N. OKAMURA, S. ISHIBASHI, and T. TAKANO
PMNL had almost the same bactericidal activity as untreated PMNL in spite of a marked reduction of phagocytosis by cytochalasin D, and that production and release of the above-mentioned bactericidal factors were marked in the cytochalasin D-treated PMNL.
Preparation of Peritoneal PMNL from Guinea Pig—PMNL was obtained from guinea pig peritoneum (13). Casein dissolved in saline to a concentration of 10% was intraperitoneally injected at a dose of 30 ml/kg in female guinea pigs of the Hartley strain to induce PMNL in the peritoneal cavity. After 18 h, the peritoneal exudate was collected and filtered through four
100 Q for 10 min. Free E. coli remained in the upper layer, whereas bacteria phagocytized by (or firmly associated with) PMNL were precipitated. Radioactivity of the precipitate was measured as an index of the phagocytizing activity of PMNL after washing the precipitate as described above. Radioactivity of the acid-soluble fraction derived from PHJDNA of E. coli was taken as an J. Biochem.
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sheets of gauze. The filtrate was centrifuged at 100 g for 5 min, and the precipitated cell pellet was washed with thymidine-supplemented Hanks solution. More than 95% of the cells thus obtained were judged to be PMNL from microscopic observations. These PMNL were resuspended in the same solution to a concentration of 1.00-1.25x10' cells/ml, and inactivated horse MATERIALS AND METHODS serum was added to a concentration of 20%; Materials—Cytochalasin D was kindly pro- this serum-supplemented PMNL suspension was vided by Shionogi Pharmaceutical Co. Escheri- used in the following experiments. chia coli 3HOT", a thymine-requiring mutant, was Measurement of Bactericidal Activity of PMNL a generous gift from Dr. S. Natori, Faculty of —One ml each of the labeled E. coli suspension Pharmaceutical Sciences, University of Tokyo. and PMNL suspension, containing almost equal Cytochrome c (horse heart, type DT), scopoletin, numbers of cells, were mixed in the following catalase (bovine liver), superoxide dismutase experiments. The mixture was incubated at (bovine blood), horse radish peroxidase, lysozyme 37°C for various periods under 95 % air-5 % CO, (egg white, grade I), and NAD were purchased with reciprocal shaking. Cytochalasin D dissolved from Sigma Chemical Co. Micrococcus lyso- in dimethylsulfoxide (5 ft\) to a concentration of deikticus (spray-dried powder) was purchased from 2 mg/ml was added to some of the mixture, while Seikagaku Kogyo Co., and 4-methyl-umbelliferyl- the same volume of dimethylsulfoxide was added 2-acetamideo-2-deoxy-jS-D-glucopyranoside and 4- to the control. The reaction was stopped by methyl-umbellyferyl-y9-glucuronide were from chilling, and 2 ml of 1% Triton X-100 in saline Koch-Light Ltd. [6-'H]Thymidine (5 Ci/mmol) was added to the incubation mixture to disrupt was a product of the Radiochemical Centre. PMNL. An aliquot (0.5 ml) of the final mixture Preparation of E. coli Labeled with [*H]- was diluted 10* times with saline, and an aliquot Thymidine—E. coli 3U0T~ was precultured in a of the diluted mixture was cultured on an agar modified M-9 medium (casamino acids 10 mg/ml, plate for 40 h to count the number of E. coli that yeast extract 0.1 mg/ml, glucose 22.6 mM, CaClj, had survived the contact with PMNL. It was 0.1 mM, MgSO4 1 mM, FeCl, 1 /IM, gelatin 10 ftgl confirmed in the preliminary experiment that ml, Na2HPO4 41 mM, KH,PO4 22.1 mM, NaCI cytochalasin D, dimethylsulfoxide, and Triton 8.5 mM, NH4C1 18.7 mM, and thymidine 8/JM), X-100 themselves had-no effect on the viability and then cultured in the same medium supple- of E. coli at the concentrations used in these exmented with 0.1 mCi of PHJthymidine. The periments. labeled E. coli was collected by centrifugation Phagocytosis and Degradation of E. coli by when the absorption at 650 nm of the cell suspen- PMNL—For the measurement of the bacteriasion reached around 0.5. The pellet was washed phagocytizing and -degrading activities of PMNL, with 10 ml of Hanks solution supplemented with the labeled E. coli and PMNL were mixed and 8 [IM thymidine and was resuspended in the same incubated as described above. After the incubasolution to a concentration of 0.8-1.7 x 107 cells/ml tion, free and phagocytized E. coli were separated (4-8 x 104 cpm/ml). This E. coli suspension was by overlaying the incubation mixture (2 ml) on used in the following experiments. 1 ml of 1 M sucrose solution and centrifuging at
BACTERICIDE BY LEUKOCYTE WITHOUT PHAGOCYTOSIS
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index of the bacteria degraded in PMNL. Two genase (18) were measured in the supernatant after ml of ice-cold 5 % trichloroacetic acid was added to centrifugation of the incubation mixture. The the above-mentioned precipitate of E. coli and ratio of the released activity to the total activity in PMNL, and the mixture was centrifuged at 1,000 g PMNL was calculated. for 10 min. Then, trichloroacetic acid (5 %, 2 ml) was added to the precipitate and the mixture was RESULTS centrifuged again after incubation at 70°C for 10 min. The radioactivities of these cold and hot Effect of Cytochalasin D on the Phagocytizing trichloroacetic acid-soluble fractions were assumed Activity of PMNL—The effect of cytochalasin D to represent the bacteria degraded by PMNL and on phagocytosis of E. coli by PMNL was examined those phagocytized but not degraded by PMNL, by incubating PITJthymidine-labeled E. coli with respectively. From the radioactivities measured, PMNL for 60 min in the presence or absence of rates of phagocytosis and degradation of the 5 ^g/ml of cytochalasin D. As mentioned in bacteria by PMNL were calculated. " MATERIALS AND METHODS," the bacteria Measurement of Superoxide Anion Production that precipitated and associated with PMNL even —Production of superoxide anions by PMNL was after washing were regarded as the phagocytized measured on the basis of reduction of cytochrome bacteria in this paper. Almost all of the bacteria c by superoxide anions (5). A mixture of E. coli were estimated to be phagocytized by the control and PMNL was incubated for 10 min in the presence PMNL, i.e. in the absence of cytochalasin D, of 50 fiM ferricytochrome c. After the incubation, since nearly 100 % of the radioactivity was recovered the mixture was centrifuged at 1,000 g for 10 min. in PMNL fraction, as shown in Fig. 1. HowThe absorption of the supernatant at 549 nm due ever, phagocytosis was inhibited by about 50% to reduced cytochrome c was read to calculate the in the cytochalasin D-treated PMNL, as shown in amount of superoxide anions produced by PMNL. the same figure. The accordance of the cytochrome c reduction with As an index of the events occurring after the superoxide anion production by PMNL was phagocytosis, degradation of DNA of E. coli was confirmed by the elimination of the reduction upon examined by measuring aH in the acid-soluble addition of 50 /Jg of superoxide dismutase to the incubation mixture. Addition of the same amount of catalase had no effect on the reduction of cytochrome c. Measurement of Hydrogen Peroxide Production —Oxidation of scopoletin was measured as an index of the production of hydrogen peroxide by PMNL {14). E. coli and PMNL were mixed and incubated for 10 min in the presence of 4 piM scopoletin and 22 nM horseradish peroxidase. The mixture was centrifuged as above after the incubation, and the intensity of the fluorescence emission of the supernatant at 460 nm was read with excitation at 350 nm. The production of hydrogen peroxide by PMNL was calculated from this value, 20 40 60 which corresponded to scopoletin oxidized by the Incubation time(min.) produced hydrogen peroxide and peroxidase. Fig. 1. Effect of cytochalasin D on the phagocytizing Enzyme Release from PMNL—Release of activity of PMNL. Radioactivity associated with enzymes from PMNL was examined during the PMNL was measured as an index of phagocytosis after incubation of PMNL with E. coli and/or cyto- the incubation of PMNL with E. coli prelabeled with chalasin D. Activities of N-acetyl-^-glucos- [•Hjthymidine either in the presence or absence of aminidase (IS), /3-glucuronidase (75), lysozyme cytochalasin D. O, 5 /
40 .
ABC MPO
-
D
n
20 ABC D
LDH
n PI
A HP
A B C D
n nnn
Fig. 4. Release of lysosomal hydrolases and lactate dehydrogenase from PMNL. Enzyme activities were measured without disruption of PMNL after the incubation for 10 min of PMNL with E. coli, cytochalasin D, or both. £-GU, 0-glucuronidase; NAGA, N-acctyl-ySglucosaminidase; MPO, myeloperoxidase; LDH, lactate dehydrogenase. A, control; B, E. coli; C, 5/ig/ml cytochalasin D ; D, E. coli and cytochalasin D.
is known to be involved in the bactericidal action of PMNL. However, the release of myeloperoxidase, which is also known to have a role in the bactericidal action, was not stimulated by either E. coli or cytochalasin D. Since the release of lactate dehydrogenase, a typical cytoplasmic marker enzyme, was not induced by either E. coli or cytochalasin D, as shown in the same figure, it is unlikely that the above-mentioned increase in the release of several lysosomal enzymes was due to decomposition of PMNL during the incubation. Figure 5 shows a marked increase in the reduction of cytochrome c on addition of cytochalasin D or E. coli, or both, during the incubation of PMNL. It was proved that the reduction was due to superoxide anions produced by PMNL, since the reduction was inhibited by superoxide dismutase but not by catalase. Since the reduction was measured with undisrupted PMNL, superoxide anions involved in the reduction were thought to be released. The amount of superoxide anions might be underestimated here, as all cytochrome c was reduced under the conditions employed in this study. Oxidation of scopoletin during the incubation of PMNL was also increased markedly by the addition of cytochalasin D or E. coli, and particularly by a combination of the two, as shown in Fig. 6. The finding that the Vol. 86, No. 2, 1979
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o u a.
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B
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C
1 2 50 n mol/
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a 20
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Fig. 5. Production and release of superoxide anions by PMNL. Reduction of cytochrome c by undisrupted PMNL was measured as an index of supcroxide anions produced and released by PMNL during incubation with E. coli, cytochalasin D, or both. A, control; B, E. coli; C, 5 /ig/ml cytochalasin D; D, E. coli and cytochalasin D.
c •5
o E _ o
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C
Fig. 6. Production and release of hydrogen peroxide by PMNL. Oxidation of scopoletin by undisrupted PMNL in the presence of horseradish peroxidase was measured as an index of hydrogen peroxide produced and released by PMNL during incubation with E. coli, cytochalasin D, or both. A, control; B, E. coli; C, 5 /ig/ml cytochalasin D; D, E. coli and cytochalasin D.
oxidation of scopoletin was inhibited by catalase, but not by superoxide dismutase, indicated that hydrogen peroxide produced by PMNL was responsible for the oxidation. This measurement was also carried out with undisrupted PMNL. The results indicate a marked increase in the
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474
N. OKAMURA, S. ISHIBASHI, and T. TAKANO
DISCUSSION As a mechanism of bactericidal action of PMNL, it is generally believed that PMNL phagocytize bacteria to form phagolysosomes (19) in which the bacteria are killed (20, 21). Reactive oxygen metabolites, Iysosomal hydrolases, and acid cationic proteins have been reported to be involved in the bactericidal action (1-10). However, the actual site where these factors play their roles in killing bacteria has not been clearly identified. In this paper, we have presented evidence that E. coli is killed by PMNL without phagocytosis in the presence of cytochalasin D. Cytochalasin D inhibited phagocytosis of the bacteria and degradation of the bacterial DNA by PMNL (Figs. 1 and 2). However, the bactericidal activity of the cytochalasin D-treated PMNL was almost the same as that of control PMNL, which phagocytized almost all the bacteria. This finding suggests that phagocytosis is not a prerequisite for the bactericidal action, at least in the case of cytochalasin D-treated PMNL (Fig. 3). Cytochalasin D itself had no toxic effect on E. coli at the concentration used. As a possible mechanism for the bactericidal action of PMNL without phagocytosis, assuming that it does exist, a marked increase in the release of superoxide anions and hydrogen peroxide was demonstrated during the incubation of PMNL
with cytochalasin D and E. coli (Figs. 5 and 6). Extracellular activity of Iysosomal enzymes was also increased, except for myeloperoxidase, after the incubation of PMNL with cytochalasin D and E. coli (Fig. 4). It is not clear why myeloperoxidase was an exception, but this finding may be related to the reported finding that this enzyme is firmly bound to the Iysosomal membrane (17). Actually, the activity was high inside PMNL. It is unlikely that the above-mentioned increase in the release of the bactericidal factors was due to the disruption of PMNL during incubation, since release of lactate dehydrogenase was not detected under the same incubation conditions (Fig. 4). Though it was found in this study that the bactericidal action of the cytochalasin D-treated PMNL seemed to occur without phagocytosis, it is unlikely that the bacteria were killed in the medium far from PMNL, since no bactericidal activity was recovered from the medium after the removal of PMNL by centrifugation. If the bactericidal factors, such as reactive oxygen metabolites, Iysosomal enzymes, etc., are released from PMNL, the site of the action of these factors may be close to the surface of PMNL where the concentration of these factors would be locally high. Such an assumption is supported by the finding the superoxide anion-generating system is associated with the cell surface of PMNL (22). It is also possible that the bacteria are killed inside PMNL but cannot be retained in the cell in the presence of cytochalasin D and are released outside the PMNL in association with the abovementioned bactericidal factors. However, since inhibition of phagocytosis of PMNL by cytochalasin E, which belongs to the same group as cytochalasin D, was clearly demonstrated electronmicroscopically (12), this seems unlikely. REFERENCES 1. Allen, R.C., Yevich, S.J., Orth, R.W., & Steelc, 2. 3. 4. 5.
R.H. (1974) Biochem. Biophys. Res. Commun. 60, 909-917 Tsan, M., Dauglass, K.H., & Mclntyre, P.A. (1977) Blood 49, 437-444 Klebanoff, S J . (1974) / . Biol. Chem. 249, 3724-3728 Baehner, R.L., Boxer, L.A., Allen, J.M., & Davis, J. (1977) Blood SO, 327-335 Babior, E.M., Kipnes, R.S., & Curnutte, J.T. (1973) / . Oin. Invest. 52, 741-744 / . Biochem.
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production and/or release of reactive oxygen metabolites from PMNL incubated in the presence of cytochalasin D and E. coli, especially of the former, and seem to account for the above-mentioned bactericidal action of cytochalasin D-treated PMNL without phagocytosis. After the incubation of PMNL with cytochalasin D and E. coli for 15 min, the incubation medium was separated by centrifugation and investigated for bactericidal activity. However, no bactericidal activity was recovered in the medium. This result may be due to excessive dilution or instability of the bactericidal factors mentioned, even if they are released. In other words, a locally high concentration of these factors on the external surface of PMNL would be necessary to kill the bacteria without phagocytosis in the presence of cytochalasin D.
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Vol. 86, No. 2, 1979
14. Root, R.K., Metcalf, J., Oshino, N., & Chance, B. (1975) / . Clin. Invest. 55, 945-955 15. Peters, T.J., Miiller, M., & de Duve, C. (1972) / . Exp. Med. 136, 1117-1139 16. Parry, R.M., Jr., Chandan, R.C., & Shahani, K.M. (1965) Proc. Soc. Exp. Biol. Med. 119, 384-386 17. Bretz, U. & Baggiolini, M. (1973) / . Cell Biol. 59, 696-707 18. Neiland, J.B. (1955) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 1, pp. 449-454, Academic Press, New York 19. Zucker-Franklin, D. & Hirsh, J.G. (1964) J. Exp. Med. 120, 569-575 20. Conn, Z.A. (1963) J. Exp. Med. 117, 27-53 21. Conn, Z.A. & Morse, S.I. (1959) /. Exp. Med. 110, 419-443 22. Goldstein, I.M., Cerqueira, M., Lind, S., & Kaplan, H.B. (1977) J. Clin. Invest. 59, 249-254
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6. Eckstein, M.R., Baehner, R.L., & Nathan, D.G. (1971) / . Clin. Invest. 50, 1985-1991 7. Johnston, R.B., Jr., Keele, B.B., Jr., Misra, H.P., Lehmeyer, J.E., Webb, L.S., Baehner, R.L., & Rajagopalan, K.V. (1975) / . Clin. Invest. 55, 13571372 8. Babior, B.M., Cumutte, J.T., & Kipnes, R.S. (1975) / . Lab. Clin. Med. 85, 235-244 9. Spitznagel, J.K. & Chi, H.Y. (1963) Am. J. Pathol. 43, 697-711 10. Zeya, H.I. & Spitznagel, J.K. (1966) / . Bacteriol. 91, 750-754 11. Nakagawara, A. & Minakami, S. (1975) Biochem. Biophys. Res. Commun. 64, 760-767 12. Nakagawara, A., Shibata, Y., Takcshige, K., & Minakami, S. (1976) Exp. Cell Res. 101, 225-234 13. Sbarra, A.J. & Karnovsky, MX. (1959) / . Biol. Chem. 234, 1355-1362
475
Evidence for Bactericidal Activity of Polymorphonuclear
Naoki OKAMURA, Sadahiko ISHLBASHI, and Tatsuya TAKANO1 Department of Physiological Chemistry, Hiroshima University School of Medicine, Kasumi, Hiroshima, Hiroshima 734 Received for publication, February 9, 1979
The relationship between phagocytosis and bactericidal action of polymorphonuclear leukocytes was examined by comparing the functions of cytochalasin D-treated leukocytes with those of the control. Measurement of phagocytotic and bacterial DNA-degrading activities using Escherichia coli prelabeled with PHJthymidine revealed that phagocytosis and bacterial DNA degradation were inhibited by treatment with cytochalasin D to about 50 and 10% of the control, respectively. Nevertheless, the bactericidal activity of the cytochalasin D-treated leukocytes was almost the same as that of the control leukocytes; almost all the bacteria were phagocytized by the latter leukocytes. Under the same experimental conditions, the production and release of superoxide anions and hydrogen peroxide, which are both known to be involved in the bactericidal action of the leukocytes, were markedly increased by cytochalasin D. Release of several lysosomal hydrolases was also increased markedly by cytochalasin D treatment, except for myeloperoxidase. However, lactate dehydrogenase, a typical cytosolic marker, was not released by the same treatment. Thus, it is unlikely that the increase in the release of the above-mentioned bactericidal factors was due to decomposition of the leukocytes. These results indicate that the site of bactericidal action of cytochalasin D-treated leukocytes is not necessarily intracellular but may be around the external surface of the cells.
In connection with the bactericidal function of polymorphonuclear leukocytes (PMNL), there is considerable interest in the involvement of reactive oxygen metabolites, such as superoxide anion, hydrogen peroxide, singlet oxygen, hydroxyl radical, etc., which are believed to be formed accompanying phagocytosis (1-8). The roles of lysosomal enzymes, such as lysozyme, cathepsins, etc., and of cationic proteins have also been con•Present address: Faculty of Pharmaceutical Sciences, Teikyo University, Tsukui, Kanagawa 199-01. Abbreviations: PMNL, polymorphonuclear leukocyte, Vol. 86, No. 2, 1979
469
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Leukocytes without Phagocytosis
sidered in the bactericidal action of PMNL (9, 10). On the other hand, it was reported that cytochalasin E as well as cytochalasin D stimulated superoxide anion production in PMNL, though both inhibited phagocytosis (11, 12). In fact, it has not yet been fully clarified how and where phagocytosis and bacteria-killing by these factors proceed in PMNL, though it is generally believed that bacteria are killed after phagocytosis and are successively digested in phagolysosomes. In this paper, we present evidence that bacteria may be killed by PMNL without phagocytosis, on the basis of findings that cytochalasin D-treated
470
N. OKAMURA, S. ISHIBASHI, and T. TAKANO
PMNL had almost the same bactericidal activity as untreated PMNL in spite of a marked reduction of phagocytosis by cytochalasin D, and that production and release of the above-mentioned bactericidal factors were marked in the cytochalasin D-treated PMNL.
Preparation of Peritoneal PMNL from Guinea Pig—PMNL was obtained from guinea pig peritoneum (13). Casein dissolved in saline to a concentration of 10% was intraperitoneally injected at a dose of 30 ml/kg in female guinea pigs of the Hartley strain to induce PMNL in the peritoneal cavity. After 18 h, the peritoneal exudate was collected and filtered through four
100 Q for 10 min. Free E. coli remained in the upper layer, whereas bacteria phagocytized by (or firmly associated with) PMNL were precipitated. Radioactivity of the precipitate was measured as an index of the phagocytizing activity of PMNL after washing the precipitate as described above. Radioactivity of the acid-soluble fraction derived from PHJDNA of E. coli was taken as an J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/86/2/469/752230 by University of Otago user on 18 January 2019
sheets of gauze. The filtrate was centrifuged at 100 g for 5 min, and the precipitated cell pellet was washed with thymidine-supplemented Hanks solution. More than 95% of the cells thus obtained were judged to be PMNL from microscopic observations. These PMNL were resuspended in the same solution to a concentration of 1.00-1.25x10' cells/ml, and inactivated horse MATERIALS AND METHODS serum was added to a concentration of 20%; Materials—Cytochalasin D was kindly pro- this serum-supplemented PMNL suspension was vided by Shionogi Pharmaceutical Co. Escheri- used in the following experiments. chia coli 3HOT", a thymine-requiring mutant, was Measurement of Bactericidal Activity of PMNL a generous gift from Dr. S. Natori, Faculty of —One ml each of the labeled E. coli suspension Pharmaceutical Sciences, University of Tokyo. and PMNL suspension, containing almost equal Cytochrome c (horse heart, type DT), scopoletin, numbers of cells, were mixed in the following catalase (bovine liver), superoxide dismutase experiments. The mixture was incubated at (bovine blood), horse radish peroxidase, lysozyme 37°C for various periods under 95 % air-5 % CO, (egg white, grade I), and NAD were purchased with reciprocal shaking. Cytochalasin D dissolved from Sigma Chemical Co. Micrococcus lyso- in dimethylsulfoxide (5 ft\) to a concentration of deikticus (spray-dried powder) was purchased from 2 mg/ml was added to some of the mixture, while Seikagaku Kogyo Co., and 4-methyl-umbelliferyl- the same volume of dimethylsulfoxide was added 2-acetamideo-2-deoxy-jS-D-glucopyranoside and 4- to the control. The reaction was stopped by methyl-umbellyferyl-y9-glucuronide were from chilling, and 2 ml of 1% Triton X-100 in saline Koch-Light Ltd. [6-'H]Thymidine (5 Ci/mmol) was added to the incubation mixture to disrupt was a product of the Radiochemical Centre. PMNL. An aliquot (0.5 ml) of the final mixture Preparation of E. coli Labeled with [*H]- was diluted 10* times with saline, and an aliquot Thymidine—E. coli 3U0T~ was precultured in a of the diluted mixture was cultured on an agar modified M-9 medium (casamino acids 10 mg/ml, plate for 40 h to count the number of E. coli that yeast extract 0.1 mg/ml, glucose 22.6 mM, CaClj, had survived the contact with PMNL. It was 0.1 mM, MgSO4 1 mM, FeCl, 1 /IM, gelatin 10 ftgl confirmed in the preliminary experiment that ml, Na2HPO4 41 mM, KH,PO4 22.1 mM, NaCI cytochalasin D, dimethylsulfoxide, and Triton 8.5 mM, NH4C1 18.7 mM, and thymidine 8/JM), X-100 themselves had-no effect on the viability and then cultured in the same medium supple- of E. coli at the concentrations used in these exmented with 0.1 mCi of PHJthymidine. The periments. labeled E. coli was collected by centrifugation Phagocytosis and Degradation of E. coli by when the absorption at 650 nm of the cell suspen- PMNL—For the measurement of the bacteriasion reached around 0.5. The pellet was washed phagocytizing and -degrading activities of PMNL, with 10 ml of Hanks solution supplemented with the labeled E. coli and PMNL were mixed and 8 [IM thymidine and was resuspended in the same incubated as described above. After the incubasolution to a concentration of 0.8-1.7 x 107 cells/ml tion, free and phagocytized E. coli were separated (4-8 x 104 cpm/ml). This E. coli suspension was by overlaying the incubation mixture (2 ml) on used in the following experiments. 1 ml of 1 M sucrose solution and centrifuging at
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index of the bacteria degraded in PMNL. Two genase (18) were measured in the supernatant after ml of ice-cold 5 % trichloroacetic acid was added to centrifugation of the incubation mixture. The the above-mentioned precipitate of E. coli and ratio of the released activity to the total activity in PMNL, and the mixture was centrifuged at 1,000 g PMNL was calculated. for 10 min. Then, trichloroacetic acid (5 %, 2 ml) was added to the precipitate and the mixture was RESULTS centrifuged again after incubation at 70°C for 10 min. The radioactivities of these cold and hot Effect of Cytochalasin D on the Phagocytizing trichloroacetic acid-soluble fractions were assumed Activity of PMNL—The effect of cytochalasin D to represent the bacteria degraded by PMNL and on phagocytosis of E. coli by PMNL was examined those phagocytized but not degraded by PMNL, by incubating PITJthymidine-labeled E. coli with respectively. From the radioactivities measured, PMNL for 60 min in the presence or absence of rates of phagocytosis and degradation of the 5 ^g/ml of cytochalasin D. As mentioned in bacteria by PMNL were calculated. " MATERIALS AND METHODS," the bacteria Measurement of Superoxide Anion Production that precipitated and associated with PMNL even —Production of superoxide anions by PMNL was after washing were regarded as the phagocytized measured on the basis of reduction of cytochrome bacteria in this paper. Almost all of the bacteria c by superoxide anions (5). A mixture of E. coli were estimated to be phagocytized by the control and PMNL was incubated for 10 min in the presence PMNL, i.e. in the absence of cytochalasin D, of 50 fiM ferricytochrome c. After the incubation, since nearly 100 % of the radioactivity was recovered the mixture was centrifuged at 1,000 g for 10 min. in PMNL fraction, as shown in Fig. 1. HowThe absorption of the supernatant at 549 nm due ever, phagocytosis was inhibited by about 50% to reduced cytochrome c was read to calculate the in the cytochalasin D-treated PMNL, as shown in amount of superoxide anions produced by PMNL. the same figure. The accordance of the cytochrome c reduction with As an index of the events occurring after the superoxide anion production by PMNL was phagocytosis, degradation of DNA of E. coli was confirmed by the elimination of the reduction upon examined by measuring aH in the acid-soluble addition of 50 /Jg of superoxide dismutase to the incubation mixture. Addition of the same amount of catalase had no effect on the reduction of cytochrome c. Measurement of Hydrogen Peroxide Production —Oxidation of scopoletin was measured as an index of the production of hydrogen peroxide by PMNL {14). E. coli and PMNL were mixed and incubated for 10 min in the presence of 4 piM scopoletin and 22 nM horseradish peroxidase. The mixture was centrifuged as above after the incubation, and the intensity of the fluorescence emission of the supernatant at 460 nm was read with excitation at 350 nm. The production of hydrogen peroxide by PMNL was calculated from this value, 20 40 60 which corresponded to scopoletin oxidized by the Incubation time(min.) produced hydrogen peroxide and peroxidase. Fig. 1. Effect of cytochalasin D on the phagocytizing Enzyme Release from PMNL—Release of activity of PMNL. Radioactivity associated with enzymes from PMNL was examined during the PMNL was measured as an index of phagocytosis after incubation of PMNL with E. coli and/or cyto- the incubation of PMNL with E. coli prelabeled with chalasin D. Activities of N-acetyl-^-glucos- [•Hjthymidine either in the presence or absence of aminidase (IS), /3-glucuronidase (75), lysozyme cytochalasin D. O, 5 /
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Fig. 4. Release of lysosomal hydrolases and lactate dehydrogenase from PMNL. Enzyme activities were measured without disruption of PMNL after the incubation for 10 min of PMNL with E. coli, cytochalasin D, or both. £-GU, 0-glucuronidase; NAGA, N-acctyl-ySglucosaminidase; MPO, myeloperoxidase; LDH, lactate dehydrogenase. A, control; B, E. coli; C, 5/ig/ml cytochalasin D ; D, E. coli and cytochalasin D.
is known to be involved in the bactericidal action of PMNL. However, the release of myeloperoxidase, which is also known to have a role in the bactericidal action, was not stimulated by either E. coli or cytochalasin D. Since the release of lactate dehydrogenase, a typical cytoplasmic marker enzyme, was not induced by either E. coli or cytochalasin D, as shown in the same figure, it is unlikely that the above-mentioned increase in the release of several lysosomal enzymes was due to decomposition of PMNL during the incubation. Figure 5 shows a marked increase in the reduction of cytochrome c on addition of cytochalasin D or E. coli, or both, during the incubation of PMNL. It was proved that the reduction was due to superoxide anions produced by PMNL, since the reduction was inhibited by superoxide dismutase but not by catalase. Since the reduction was measured with undisrupted PMNL, superoxide anions involved in the reduction were thought to be released. The amount of superoxide anions might be underestimated here, as all cytochrome c was reduced under the conditions employed in this study. Oxidation of scopoletin during the incubation of PMNL was also increased markedly by the addition of cytochalasin D or E. coli, and particularly by a combination of the two, as shown in Fig. 6. The finding that the Vol. 86, No. 2, 1979
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Fig. 5. Production and release of superoxide anions by PMNL. Reduction of cytochrome c by undisrupted PMNL was measured as an index of supcroxide anions produced and released by PMNL during incubation with E. coli, cytochalasin D, or both. A, control; B, E. coli; C, 5 /ig/ml cytochalasin D; D, E. coli and cytochalasin D.
c •5
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Fig. 6. Production and release of hydrogen peroxide by PMNL. Oxidation of scopoletin by undisrupted PMNL in the presence of horseradish peroxidase was measured as an index of hydrogen peroxide produced and released by PMNL during incubation with E. coli, cytochalasin D, or both. A, control; B, E. coli; C, 5 /ig/ml cytochalasin D; D, E. coli and cytochalasin D.
oxidation of scopoletin was inhibited by catalase, but not by superoxide dismutase, indicated that hydrogen peroxide produced by PMNL was responsible for the oxidation. This measurement was also carried out with undisrupted PMNL. The results indicate a marked increase in the
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N. OKAMURA, S. ISHIBASHI, and T. TAKANO
DISCUSSION As a mechanism of bactericidal action of PMNL, it is generally believed that PMNL phagocytize bacteria to form phagolysosomes (19) in which the bacteria are killed (20, 21). Reactive oxygen metabolites, Iysosomal hydrolases, and acid cationic proteins have been reported to be involved in the bactericidal action (1-10). However, the actual site where these factors play their roles in killing bacteria has not been clearly identified. In this paper, we have presented evidence that E. coli is killed by PMNL without phagocytosis in the presence of cytochalasin D. Cytochalasin D inhibited phagocytosis of the bacteria and degradation of the bacterial DNA by PMNL (Figs. 1 and 2). However, the bactericidal activity of the cytochalasin D-treated PMNL was almost the same as that of control PMNL, which phagocytized almost all the bacteria. This finding suggests that phagocytosis is not a prerequisite for the bactericidal action, at least in the case of cytochalasin D-treated PMNL (Fig. 3). Cytochalasin D itself had no toxic effect on E. coli at the concentration used. As a possible mechanism for the bactericidal action of PMNL without phagocytosis, assuming that it does exist, a marked increase in the release of superoxide anions and hydrogen peroxide was demonstrated during the incubation of PMNL
with cytochalasin D and E. coli (Figs. 5 and 6). Extracellular activity of Iysosomal enzymes was also increased, except for myeloperoxidase, after the incubation of PMNL with cytochalasin D and E. coli (Fig. 4). It is not clear why myeloperoxidase was an exception, but this finding may be related to the reported finding that this enzyme is firmly bound to the Iysosomal membrane (17). Actually, the activity was high inside PMNL. It is unlikely that the above-mentioned increase in the release of the bactericidal factors was due to the disruption of PMNL during incubation, since release of lactate dehydrogenase was not detected under the same incubation conditions (Fig. 4). Though it was found in this study that the bactericidal action of the cytochalasin D-treated PMNL seemed to occur without phagocytosis, it is unlikely that the bacteria were killed in the medium far from PMNL, since no bactericidal activity was recovered from the medium after the removal of PMNL by centrifugation. If the bactericidal factors, such as reactive oxygen metabolites, Iysosomal enzymes, etc., are released from PMNL, the site of the action of these factors may be close to the surface of PMNL where the concentration of these factors would be locally high. Such an assumption is supported by the finding the superoxide anion-generating system is associated with the cell surface of PMNL (22). It is also possible that the bacteria are killed inside PMNL but cannot be retained in the cell in the presence of cytochalasin D and are released outside the PMNL in association with the abovementioned bactericidal factors. However, since inhibition of phagocytosis of PMNL by cytochalasin E, which belongs to the same group as cytochalasin D, was clearly demonstrated electronmicroscopically (12), this seems unlikely. REFERENCES 1. Allen, R.C., Yevich, S.J., Orth, R.W., & Steelc, 2. 3. 4. 5.
R.H. (1974) Biochem. Biophys. Res. Commun. 60, 909-917 Tsan, M., Dauglass, K.H., & Mclntyre, P.A. (1977) Blood 49, 437-444 Klebanoff, S J . (1974) / . Biol. Chem. 249, 3724-3728 Baehner, R.L., Boxer, L.A., Allen, J.M., & Davis, J. (1977) Blood SO, 327-335 Babior, E.M., Kipnes, R.S., & Curnutte, J.T. (1973) / . Oin. Invest. 52, 741-744 / . Biochem.
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production and/or release of reactive oxygen metabolites from PMNL incubated in the presence of cytochalasin D and E. coli, especially of the former, and seem to account for the above-mentioned bactericidal action of cytochalasin D-treated PMNL without phagocytosis. After the incubation of PMNL with cytochalasin D and E. coli for 15 min, the incubation medium was separated by centrifugation and investigated for bactericidal activity. However, no bactericidal activity was recovered in the medium. This result may be due to excessive dilution or instability of the bactericidal factors mentioned, even if they are released. In other words, a locally high concentration of these factors on the external surface of PMNL would be necessary to kill the bacteria without phagocytosis in the presence of cytochalasin D.
BACTERiaDE BY LEUKOCYTE WITHOUT PHAGOCYTOSIS
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14. Root, R.K., Metcalf, J., Oshino, N., & Chance, B. (1975) / . Clin. Invest. 55, 945-955 15. Peters, T.J., Miiller, M., & de Duve, C. (1972) / . Exp. Med. 136, 1117-1139 16. Parry, R.M., Jr., Chandan, R.C., & Shahani, K.M. (1965) Proc. Soc. Exp. Biol. Med. 119, 384-386 17. Bretz, U. & Baggiolini, M. (1973) / . Cell Biol. 59, 696-707 18. Neiland, J.B. (1955) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 1, pp. 449-454, Academic Press, New York 19. Zucker-Franklin, D. & Hirsh, J.G. (1964) J. Exp. Med. 120, 569-575 20. Conn, Z.A. (1963) J. Exp. Med. 117, 27-53 21. Conn, Z.A. & Morse, S.I. (1959) /. Exp. Med. 110, 419-443 22. Goldstein, I.M., Cerqueira, M., Lind, S., & Kaplan, H.B. (1977) J. Clin. Invest. 59, 249-254
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6. Eckstein, M.R., Baehner, R.L., & Nathan, D.G. (1971) / . Clin. Invest. 50, 1985-1991 7. Johnston, R.B., Jr., Keele, B.B., Jr., Misra, H.P., Lehmeyer, J.E., Webb, L.S., Baehner, R.L., & Rajagopalan, K.V. (1975) / . Clin. Invest. 55, 13571372 8. Babior, B.M., Cumutte, J.T., & Kipnes, R.S. (1975) / . Lab. Clin. Med. 85, 235-244 9. Spitznagel, J.K. & Chi, H.Y. (1963) Am. J. Pathol. 43, 697-711 10. Zeya, H.I. & Spitznagel, J.K. (1966) / . Bacteriol. 91, 750-754 11. Nakagawara, A. & Minakami, S. (1975) Biochem. Biophys. Res. Commun. 64, 760-767 12. Nakagawara, A., Shibata, Y., Takcshige, K., & Minakami, S. (1976) Exp. Cell Res. 101, 225-234 13. Sbarra, A.J. & Karnovsky, MX. (1959) / . Biol. Chem. 234, 1355-1362
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