/ . Biochtm., 77, 559-566 (1975)

Metabolic Pattern of Polymorphonuclear Leucocytes Induced by Trypsin-digested Microsomes1 Koichiro TAKESHIGE,* Akira NAKAGAWARA,* Tanenori HATAE,** and Shigeki MINAKAMI* • Department of Biochemistry and *• Department of Anatomy, Kyushu University School of Medicine, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, August 17, 1974

The addition of trypsinfEC 3.4.21.4]-digested liver microsomes induced cyanideinsensitive respiration in guinea pig polymorphonuclear leucocytes with concomitant acceleration of the hexose monophosphate oxidative pathway. The respiration was insensitive to inhibitors of mitochondrial respiration but sensitive to glycolytic inhibitors. These metabolic alterations are similar to those associated with phagocytosis, though the digested microsomes were apparently not taken up by the cells and probably trigger the metabolic changes by interaction with the cellular membrane. Intact microsomes or microsomes treated with chymotrypsin [EC 3.4.21.1], bacterial proteinase, ribonuclease [EC 3.1.4.22], or neuraminidase [EC 3.2.1.18] could not induce such respiration.

polymorphonuclear leucocytes in the presence of rat liver particles, we found that trypsintreated microsomes induced cyanide-insensitive respiration without being ingested into the cells. The present paper is about the metabolic pattern of polymorphonuclear leucocytes induced by the trypsin-digested microsomes as well as on the modification in the microsomes which causes such respiration.

Phagocytosis in polymorphonuclear leucocytes is accompanied by the appearance of cyanideinsensitive respiration and the stimulation of the hexose monophosphate oxidative pathway (/, 2). They are considered to be the metabolic processes triggered by the interaction of the cellular membrane with particles and play an important role in the intracellular killing of bacteria. Similar metabolic alterations have been observed when certain agents such as endotoxin (3), antileucocyte antibodies (4), surface active agents (3, 5, 6), and cytochalasin E (7) interacted with the cells. During the study on the metabolism of

EXPERIMENTAL PROCEDURE

1

This study was supported in part by the Yamanouchi Foundation of Metabolism and Diseases and by a Research Grant from the Ministry of Education. Vol. 77, No. 3, 1975

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Cell Preparations — Leucocytes were obtained from guinea pig peritoneal exudates 16 to 18 hr after intraperitoneal injection of 2% sterilized solution of sodium caseinate (8). The cells were then washed out of the peritoneal cavity with ice-cold 0.85% NaCl solu-

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K. TAKESHIGE, A. NAKAGAWARA, T. HATAE, and S. MINAKAMI

tion, centrifuged at 80 xg for 10 min and suspended in the saline solution. The cells were kept in an ice-bucket before use. Ehrlich ascites tumor cells were obtained from the male mice which had been transplanted intraperitoneally 10 days before. The contamination of leucocytes was about 2%. Preparation of Digested Microsomes—Microsomes were prepared by centrifugation of liver homogenates in 0.25 M sucrose solution between 10,000X0 for 10 min and 105,000xg for 60 min. Male Wistar rats weighing 150— 200 g were used. To a microsomal suspension (10 mg/ml) in a 20 mM K,HPO«-KH2PO4 buffer of pH 7.4, 10 fig of trypsin per mg of microsomal protein was added and the suspension was kept at 4° for 12 hr. The digestion was stopped by the addition of soybean trypsin-inhibitor in a quantity 3 times that of trypsin used. The digested microsomes were centrifuged at 105,000 x g for 60 min and the pellet was suspended in 50 mM Tris-maleate buffer, pH 7.4. Centrifugation and resuspension were carried out to wash the particles. Digestion of microsomes with other enzymes was carried out with chymotrypsin [EC 3.4.21.1] (100 fig), nagarse (20 fig), phospholipase A [EC 3.1.1. 4] (100 fig), phospholipase C [EC 3.1.4.3] (100 fig), and pancreatic ribonuclease [EC 3.1.4.22] (5 /ig) for mg microsomal protein, at 4° for 12 hr. One unit of neuraminidase [EC 3.2.1.18] was added to the microsomes for each 3 mg of microsomal protein and incubated at 37° for 2 hr. Subfradionation of Microsomes—Rough and smooth subfractions of microsomes were separated according to the method of Omura and Kuriyama (9). Two male Wistar rats, fasted for 24 hr, were sacrificed and the livers were homogenized with 3 volumes of 30% sucrose solution containing 0.5 mM MgCl2 without perfusion. The homogenate was centrifuged at 24,000X0 for 20 min and the supernatant was overlaid on 4 ml of 45% sucrose solution containing 0.5 mM MgCl*. After centrifugation at 97,000xg for 8 hr,, the smooth microsomal fraction found at the interface of two sucrose layers was diluted with 2 volumes of water and the rough microsomal fraction at

the bottom of the tube was suspended in 0.25 M sucrose solution. Each subtraction was centrifuged at 78,000xg for 60 min. The pellets were washed with 0.15 M KC1 and resuspended in 0.25M sucrose solution. Oxygen Uptake—Respiration was measured by a Clark-type oxygen electrode (Yellow Spring Instrument Co., Ohio) in a closed vessel at 37°. Approximately 7xlO 7 cells were suspended in 3.0 ml of Krebs Ringer phosphate buffer, pH 7.4, in which the CaCl2 concentration was modified to 0.9 mM. Estimation of the Hexose Monophosphate Oxidative Pathway—A cell suspension in the Ringer phosphate buffer, containing 0.5 ^Ci of [1-14C] or [6-uC]glucose (6^moles), was incubated in a manometric flask at 37° with shaking at 60 strokes per min. The final volume of the reaction mixture was 1.2 ml and the reaction was started by the addition of particles and stopped by the addition of 0.1 ml of 100 g/dl trichloroacetic acid. Radioactive carbon dioxide was absorbed into 0.1 ml 1 N KOH in the centre well by further incubation for 1 hr. The radioactivity in the KOH solution was measured by a Hitachi-Horiba liquid scintillation spectrometer LS-500 using a toluene scintillator containing Triton X-100 (70). Analytical Procedures—Protein was determined according to the method of Lowry et al. (11) with the use of bovine serum albumin as a standard. Phospholipid was analyzed by a modification of the Schneider method (12). The perchloric acid precipitate was extracted with ethanol and then with ethanol-diethyl ether (3 : 1, v/v). The combinded extracts were digested with sulfuric acid and phosphorus was measured by the method of Fiske and SubbaRow (13). Cytochrome bs was determined from the dithionite-reduced minus oxidized difference spectrum. NADPH-cytochrome c reductase [EC 1.6.2.4] activity was measured according to the method of Dallner et al. (14). Electron Microscopy-^-CeWs were fixed in 3% glutaraldehyde in a 0.1 M phosphate buffer, pH 7.4, and post-fixed in 1% osmium tetroxide in the same buffer. Thin sections were double stained with uranyl acetate (15) and lead citrate (16). Electron micrographs were taken / . Biochem.

METABOLIC ALTERATIONS IN LEUCOCYTES

using a Hitachi HU-12 microscope with an accelerating voltage of 100 kV. Reagents—Trypsin(Type HI), chymotrypsin (Type II), phospholipase A {Vipera russellt), phospholipase C(Type I), and pancreatic ribonuclease(Type III-A) were obtained from Sigma Chem. Co., U.S.A. Trypsin inhibitor (soybean) was obtained from Bohringer u. So'hne, Mannheim, Germany and nagarse from the Nagase Sangyo Co., Japan. D-[6-14C] and [l-14C]glucose were obtained from the Daiichi Pure Chem. Co., Japan. RESULTS Stimulation of Respiration—When trypsintreated microsomes were added to polymorphonuclear leucocytes, a dramatic increase in respiration appeared without an appreciable lag time (Fig. 1A). The induced respiration was insensitive to cyanide and the addition of the digested microsomes in the presence of cyanide showed two phases of respiration, consisting firstly of rapid oxygen uptake, which continued for about 2 min and secondly a slow respiratory phase which continued for more than 10 min (Fig. IB). The rate of the initial phase increased with the increase in the amount of the digested microsomes added, with saturation at about 2 mg (Fig. 1C) and further addition in the second phase could not induce another burst of respiration, though the addition of particles such as heat-killed E. coli or polystyrene latex beads stimulated respiration (Fig. ID). The stimulation seemed to be related neither to substances released from the microsomal membrane nor to any enzyme unmasked by the digestion, because repeated washing, prolonged dialysis or heat treatment of the preparation at 80° for * 15 mm did not abolish the stimulating effect. No stimulation was observed in polymorphonuclear leucocytes with untreated microsomes nor in Ehrlich ascites tumor cells with the digested microsomes. The stimulation of respiration could not be attributed to the action of trypsin on the cells since digestion was terminated by the addition of an excess of trypsin inhibitor and the microsomes were washed. The addition of trypsin to the cells with or without unVol. 77, No. 3, 1975

561

treated microsomes did not cause any change in respiration but the addition of digested microsomes in this condition induced respiratory change (Fig. IE). Inhibitors—The respiratory stimulation by the digested microsomes was not only insensitive to cyanide but also insensitive to other inhibitors of mitochondrial respiration such as rotenone and antimycin A. It could be inhibited by the inhibitors of glycolysis such as monoiodoacetate or 2-deoxyglucose (Fig. IF). Stimulation of the Hexose Monophosphate Oxidative Pathway—The production of 14COi from [1-14C] and [6-uC]glucose in polymorphonuclear leucocytes in the presence of trypsindigested microsomes was shown in Fig. 2A. The increased formation of UCO2 from [1-14C]glucose was observed without an appreciable increase of u CO f from [6-14C]glucose, indicating the stimulation of the hexose monophosphate oxidative pathway, in comparison with the control experiments without microsomes or with untreated microsomes (Fig. 2B). The time-course of 14CO2 formation appeared to be in accord with the respiratory stimulation: initial rapid 14COj formation was followed by slow formation. This is in contrast to that associated with phagocytosis which showed a lag time and a gradual increase in 14CO2 formation (Fig. 2C). Electron Microscopic Observation—No ingestion of the microsomes was observed electron microscopically when leucocytes were incubated with trypsin-digested microsomes (Fig. 3), while in the control experiment with the same batch of cells incubated with heat-killed E. coli, formation of phagosomes and phagolysosomes was observed. The search for the change in the cellular membrane due to contact with the digested microsomes was unsuccessful. Effect of Microsomes Treated with Other Enzymes—The oxygen uptake of polymorphonuclear leucocytes in the presence of microsomes treated with other hydrolytic enzymes was studied. Microsomes treated with phospholipase A and phospholipase C induced cyanide-insensitive respiration in polymorphonuclear leucocytes but the respiration appeared with a significant lag time and did not switch

562

K. TAKESHIGE, A. NAKAGAWARA, T. HATAE, and S. MINAKAMI Try-Ms 1.5mg

D

Try-Ms 1.5mg

4 Glu 5mM KCNImM

i. Try-Ms 1.0mg T PS10.5mg

AOJ1MO2

T

•| 3 min 5min

•Oj-0

•02 =

KCN imM

Try-Ms 1.5mg

Try-Ms 1.«mg

^1 Glu 5mM

t

t

4.

Trypsin OJmd Untreated Ms 1.3mg

4OuMO2

T -•I 5 mln |

5 mln

•02-0

•02-0

KCNImM Glu SmM y MlA2mM

1mM

DOGIOmM

t

^f Try-Ms 1.5 mg

Try-Ms 1.5 mg

• | 10 min

Fig. 1. Effect of trypsin-digested microsomes (Try-Ms) on the respiration of polymorphonuclear leucocytes. Oxygen-electrode traces with a Clark oxygen electrode in a closed vessel at 37°. The cells were suspended in 3.0 ml of modified Krebs Ringer phosphate buffer (CaCli 0.9 mM) with 5 mM glucose (Glu). The amount of trypsin-digested microsomes and number of cells were 1.5 mg protein and 7x10', respectively, unless otherwise stated. (A) Stimulation of respiration by trypsin-treated microsomes. (B) Time course of respiratory stimulation in the presence of cyanide (KCN). (C) Effect of varying amounts of the digested microsomes. The number of cells was 4xlO 7 . (D) Repeated additions of the digested microsomes (1.5 mg and 1.0 mg) and polystyrene latex beads (PS, 10.5 mg dry weight). (E) Pretreatment of the cells with trypsin (100 fg) before the addition of intact (1.3 mg) and digested (1.4 mg) microsomes. (F) Effect of glycolytic inhibitors (monoiodoacetate-(MIA) and deoxyglucose (DOG)) on respiration. In the experiments with deoxyglucose, glucose was omitted.

/ . Biochem.

METABOLIC ALTERATIONS IN LEUCOCYTES

4

6

Time ( Min )

8

I

563

» 6 6 Tim* ( Hln )

2

( 6 Tim* ( Hln )

Fig. 2. "COs formation from glucose in polymorphonuclear leucocytes. The cells (4X107) were incubated with 0.5 fid of [l-'KI] or [6-14C]glucose (6 //moles). The radioactivity from the former is represented by circles and that from the latter by triangles. Particles were added at the start of the experiments and the results are expressed by closed symbols. Control experiments without the addition of particles are expressed by open symbols. (A) Effect of trypsin-digested microsomes (0.7 rag). (B) Effect of untreated microsomes (0.7 mg). (C) Effect of heat-killed E. coli (0.3 mg dry weight).

to the slow phase as observed with trypsindigested microsomes. Treatment with other enzymes, namely chymotrypsin, nagarse (bacterial protease), pancreatic ribonuclease, and neuraminidase, did not stimulate respiration. It seems necessary to point out the special property of trypsin among proteases to modify the microsomal membrane to interact with the cellular membrane. Chymotrypsin, a combination of chymotrypsin and trypsin (Fig. 4A) or nagarse could not modify the microsomes to stimulate respiration. Trypsin and chymotrypsin released proteins from microsomes essentially to the same extent as shown in Fig. 5. Subfradionation - of -Microsomes—The -mi— crosomes were further fractionated into rough and smooth subfractions. A marked stimulation of respiration was observed when trypsindigested smooth microsomes were added to the cells, in contrast to the slight increase of respiration with trypsin-digested rough microsomes (Fig. 4B). Without digestion, neither fraction stimulated respiration. Other cellular particles such as mitochondria did not stimulate respiration even after treatment with trypsin. Vol. 77, No. 3, 1975

DISCUSSION

The appearance of cyanide-insensitive respiration with concomitant stimulation of the hexose monophosphate oxidative pathway is the characteristic of polymorphonuclear leucocytes during phagocytosis. A similar phenomenon was observed when the cells were treated with detergents (3, 5, 6) and phospholipase (17) which may affect the membrane structure of the cellular surface. The metabolic pattern of polymorphonuclear leucocytes in the presence of trypsin-treated microsomes is similar to the above mentioned metabolic alteration: -i)- respiration was- insensitive tocyanide^anti-~ mycin A, and rotenone, 2) the respiration could be inhibited by glycolytic inhibitors such as monoiodoacetate and 2-deoxyglucose, and 3) acceleration of 14COi formation from [l-uC]glucose. The time course of the respiration, however, was different from that of phagocytosis. The respiration induced by the digested microsomes appeared without an appreciable lag time and rapid oxygen consumption, which continued only for about 2 min, was followed by slow respiration. Another difference to be

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K. TAKESHIGE, A. NAKAGAWARA, T. HATAE, and S. MlNAKAMI

Fig. 3. Electron micrograph of polymorphonuclear leucocytes incubated with trypsin-digested microsomes for 20 min. (A) X 13,000. (B) x64,000. No ingestion of the microsomes was observed, though the cells extended pseudopodia when in contact with trypsin-digested microsomes (shown by arrows). No changes in the lysosomes or in the unit membrane structure of the plasma membrane of the pseudopodia could be found.

/ . Biochem.

METABOLIC ALTERATIONS IN LEUCOCYTES KCNImM

565

Try-Ms 1.5mg

I

Glu 5mM f [Try-Chym]-Ms 1.5 mg [Chym-Try]-Ms 1.5 mg

5min • 02=0

B

- y ^

2.5

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Try-smooth Ms / 1.5 mg

Try-rough Ms Glu 5mMf / 1.5 mg KCNImM Glu 5mM | KCNImM

• I 3 min |* 25

50

75

100

yg Chymotrypsin / mg Microsomes

Fig. 4. Effect of microsomal preparations on respiration. (A) Addition of microsomes first digested with trypsin and then with chymotrypsin ([Try-Chym]Ms, 1.5 mg) and those first digested with chymotrypsin and then with trypsin ([Chym-Try]-Ms, 1.5 mg). (B) Addition of a smooth microsomal fraction digested with trypsin (Try-smooth Ms, 1.5 mg) and a rough microsomal fraction digested with trypsin (Try-rough Ms, 1.5 mg). The conditions were as described in the legends for Fig. 1.

Fig. 5. Proteolytic digestion of microsomes with various amounts of trypsin (A) and chymotrypsin (B). Microsomes (10 mg protein/ml in 0.05 M Tris-HCl buffer, pH 7.4) were digested with the indicated amounts of trypsin and chymotrypsin at 0° for 15 hr. The amounts found in the supernatant of 105,000 X g for 60 min were expressed as a percentage of the digested sample. O, NADPH cytochrome c reductase; • , protein; x, cytochrome bs; A, phospholipid.

noted is that the respiratory increments induced both by phagocytosis of E. colt and by trypsin-digested microsomes are inhibited by dibutyryl cyclic AMP, while with dibutyryl cyclic GMP the latter is inhibited and the former remains unchanged (subsequent paper

released by the treatment of microsomes with trypsin to the intracellular metabolic system could be eliminated because neither repeated washing nor prolonged dialysis of the digested microsomes reduced the capacity to induce cyanide-insensitive respiration. In connection with the interaction of particles with the cellular membrane, it may be necessary to consider the membrane structure of particles which induce cyanide-insensitive respiration. Microsomes treated with trypsin activate respiration, while intact microsomes or the ones treated with either chymotrypsin or a combination of trypsin and chymotrypsin

(18)). The metabolic change induced by trypsindigested microsomes may be ascribed to the interaction of the particles with the cellular membrane, since neither ingestion of particles nor the formation of phagosomes was observed by electron microscopy. Further, the possibility of direct action by any soluble substance .Vol. 77, No. 3, 1975

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could not activate respiration, though trypsin and chymotrypsin released proteins to essentially the same extent. Bacterial proteinase was also ineffective. Microsomes treated with phospholipase A or phospholipase C induced cyanide-insensitive respiration but the time course was significantly different from that observed with trypsin - digested microsomes. Trypsin seems to be unique in unmasking some region of the microsomal surface which reacts with the cellular membrane to trigger metabolic changes. REFERENCES 1. Sbarra, A.J. & Karnovsky, M.L. (1959) / . Biol. Chem. 234, 1355-1362 2. Iyer, G.Y.N., Islam, M.F., & Quastel, J.H. (1961) Nature 192, 535-541 3. Graham, R.C., Karnovsky, M.J., Shaffer, A.W., Glase, E.A., & Karnovsky, M.L. (1967) / . Cell Biol. 32, 629-647 4. Rossi, F., Zatti, M., Patriarca, P., & Cramer, R. (1970) Experientia 26, 491-492 5. Zatti, M. & Rossi, F. (1967) Biochim. Biophys. Ada 148, 553-555

6. Kakinuma, K. (1974) Biochim. Biophys. Ada 348, 76-85 7. Nakagawara, A., Takeshige, K., & Minakami, S. (1974) Exptl. Cell Res. 87, 392-394 8. Kakinuma, K. (1968) Jap. J. Exp. Med. 38, 165169 9. Omura, T. & Kuriyama, Y. (1971) / . Biochem. 69, 651-658 10. Patterson, M.S. & Greeme, R.C. (1965) Anal. Chem. 37, 854-857 11. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265275 12. Schneider, W.C. (1945) / . Biol. Chem. 161, 293303 13. Fiske, C.H. & SubbaRow, Y. (1925) / . Biol. Chem. 66, 375-400 14. Dallner, G., Siekevitz, P., & Palade, G.E. (1966) / . Cell Biol. 30, 97-117 15. Watson, M.L. (1958) / . Biophys. Biochem. Cytol. 4, 475-478 16. Reynolds, F.S. (1963) / . Cell Biol. 17, 208-212 17. Patriarca, P., Zatti, M., Cramer, R., & Rossi, F. (1970) Life Sciences 9, 841-849 18. Nakagawara, A., Takeshige, K., & Minakami, 5. (1974) / . Biochem. 77, 567-573

/ . Biochem.