Phagocytosis by human blood leucocytes during suppression of glycolysis

Experimental

PHAGOCYTOSIS

Cell Research 56 (1969) 15-23

BY HUMAN BLOOD LEUCOCYTES SUPPRESSION OF GLYCOLYSIS PHYLLIS

BODEL

DURING

and S. E. MALAWISTA

Department of Internal Medicine, Yale University School of Medicine, New Haven, Corm. 06510, USA

SUMMARY

Phagocytosis of staphylococciby humanbloodleucoeytes wasmeasured duringsuppression of glycolyaiswith iodoacetate,2 x lO-4M, and fluoride,2 x lo-* M. Both inhibitorapreventedthe increased respirationusuallyassociated with phagocytosis. When disappearance of superuatant bacteriawasmeasured, leucocyteapretreatedwith iodoacetateremovedasmanybacteriaasdid normalleucocytea, indicatingno alterationof phagoeyticfunctionwith thisinhibitor. In contrast, leucocytespretreatedwith sodiumfluorideshowedmarkedinhibitionof phagocytosis. Cellspretreatedwith iodoacetatewereobservedto have lessgranulelysisafter phagocytosis thannormalleucoeytea. Measurements of granule-associated acidphosphataae activity confirmed this observation. Cellstreatedwith sodiumfluoride becameswollen,vacuolated,Permeable to eosindye, and wererarely phagocytic.In contrast,suchchangeswerenot observedwith iodoaeetatein concentrationsup to 5 x lo-* M. Thusfluorideappearsto havea greatereffect on cellfunctionthan canbe attributedto glycolytic blockadealone. Rapid lossof acid-soluble ATP from blood leucocytesoccurredafter phagocytosis, and was accentuated in the presence of iodoacetate. Theseresultsindicatethat activeglycolysisis not essential for phagocytosis by humanblood leucocytes.They suggest that somepre-formedenergysourcemay be availableto thesecellsfor thisfunction.

Phagocytosis is a basic physiological function of many cells, including polymorphonuclear leucocytes. Although this processhas been studied for many years, the changes in cellular activity which result in the ingestion of a particle are still not clearly understood. Several investigators have reported that certain biochemical changes accompany phagocytosis [12, 13, l&23,26]. For polymorphonuclear leucocytes these changes include increased glycolysis, depletion of glycogen, increased turnover of certain lipids, and a marked increase in oxygen consumption by way of the hexose monophosphate shunt [20]. Human [I] and guinea pig [23] polymorphonuclear leucocytes derive almost all their energy from the glycolytic pathway. The increased activity of this pathway which normally occurs with phagocytosis can be prevented by iodoacetate or fluoride, and phagocytosis is re-

portedly inhibited. On the ‘other hand, addition of cyanide or dinitrophenol, agents that block cytochrome-linked oxidation, apparently does not affect the numbers of particles ingested. Similarly, phagocytosis proceeds normally under anaerobic conditions [2, 12, 23, 241. The present studies were undertaken to investigate the dependence of phagocytosis on glycolysis. Sodium iodoacetate and sodium fluoride were used as inhibitors. Our results indicate that normal phagocytosis can occur in human blood leucocytes when glycolysis is suppressed.

MATERIALS

AND METHODS

Methodsfor preparationand incubationof leuwcytea from humanblood, and quantificationof phagocytosis, have beendescribed previously(211. Briefly, 2 vol of 3% dextran in salinewere addedto about 100ml of heExptl Cell Res 56

16 Phyllis Bode1 & S. E. Malawista

A

6

Time, mitt; ordinate: % remaining in supernatant. (A) Multiplicity 10: 1; (E) multiplicity 23 : 1. Fig. 1. Average numbers of live staphylococci (STAPH) remaining in flask supernatants after phagocytosis by leucocytes (WBC), preincubated in buffer, iodoacetate (IAA) 2 x IO-’ M, or fluoride (F) 2 x IO-* M. Standard errors of the mean are indicated. Multiplicity refers to numbers of bacteria per polymorphonuclear leucocyte. Average results of 10 expts, 4 on the left, 6 on the right. Values for staphylococci alone are averages of samples from flasks with and without iodoacetate and fluoride. Abscissa:

parinized blood and the leucocyte-rich supernatant removed. The cells were washed with a Krebs-Ringer phosphate (KRP) buffer, and most red cells removed by hypotonic lysis. The final leucocyte suspension, containing about 3 x 10’ WBC/ml, was incubated in a 12% serum KRP buffer medium, with or without inhibitors and bacteria. Incubation was carried out in Warburg flasks containing a total volume of 2.8 ml, or in 25 ml Erlenmeyer flasks containing exactly two or three times this volume. Control flasks in each experiment contained bacteria in serum-buffer, with and without inhibitors. Sodium iodoacetate (Eastman Organic Chem.. Rochester), and sodium fluoride were each dissolved’in saline, filtered through a UF sintered-glass filter. and stored at -20°C. In a-few experiments a similar preparation of iodoacetic acid was used. As described previously [21], live alpha-hemolytic streptococci, Staphylococcus albus or Staphylococcus aureus S02A [17] were used to measure phagocytosis. Live bacteria remaining both in the flask supernatant and within the cells were estimated by a modification of the method of Cohn & Morse [I I]. For studies of respiration. lactate production and granule lysis, heat-killed- Staphylococcus albus or aureus were prepared by autoclaving a suspension of bacteria in saline for 20 min. Numbers of bacteria were estimated by transmittance at 600 m,u. Preparation of granule and supernatant fractions from leucocytes, and estimation of granule-associated acid phosphatase were carried out by the methods of Cohn & Hirsch, and Shinowara, Jones & Reinhart, as described previolusly 1211. Lactate concentrations in the incubation supernatant were measured bv conversion of DPN in the presence of lactic dehydrogenase [3]. Reagents were obtained from C. F. Boehringer and Soehne. Mannhein. Germany. Preparation of thin coverslip smears for ob: servation of cell morphology has been described previously [21]. In some experiments cells in suspension were fixed in an equal volume of 2 % buffered glutaraldehyde. After centrifugation, the cell buttons were overlaid with Exptl

Cell

Res 56

the original fixative, post-fixed in OsO,, and embedded in Maraglas. One-micron sections, stained with Mallory’s Azur II and methylene blue, were examined by light microscopy. Eosin counts were done by the method of Hanks St Wallace [14]. For measurement of ATP, leucocytes were prepared as usual from blood, or washed with 200 ml heparinized saline from rabbit peritoneal exudates, harvested four hours after intraperitoneal infusion of 200 ml 0.1 96 glucogen. Cells were-suspended in 10 ml of 12-15 % serum-KRP buffer. and divided into aliauots of about 1 x IO* cells. One al&tot was placed immediately in ice. Others were incubated on a Dubnoff shaker, alone, with heat-killed Staphylococcus albus 30 : 1, or with iodoacetate 2 x 1O-4 M with or without bacteria. After incubation, samples were removed at once to ice, centrifuged at 420 g at 4°C. for 10 min, and the supernatant discarded. One ml of ice-cold saline was added to the cell button, and the suspension was then homogenized in ice for 1 min. One ml 0.6 M perchloric acid was then added, the suspension was centrifuged at 1500 g for 10 min, and the supernatant assayed for ATP activity by the UV test employing the phosphoglucokinase reaction [4]. Reagents were obtained from Boehringer. A standard curve was first constructed for the assay of small amounts of ATP. A plot of E:hrnmp was linear for concentrations between 0.02 and 0.22 mg ATP/ml. Readings which gave values less than 0.02 mg were arbitrarily called 0.01 for computation of averages.

RESULTS Effects of iodoacetate and fluoride on phagocytosis of staphylococci

White blood cells were incubated for one hour with and without iodoacetate (2 or 4 x 1O-4 M) or sodium fluoride (2 x 1O-2 M). Live staphylococci were then added to the flasks and duplicate samples removed 20 and ‘40 min afterwards. Control flasks containing bacteria in serum-buffer medium with or without inhibitors were sampled at 40 min. The results of ten experiments are presented in fig. 1. Percentage of bacteria removed from the supernatant is plotted as a measure of phagocytosis. It is apparent that iodoacetate had no effect on phagocytosis (p= )0.5), either in the experiments shown on the left, in which the multiplicity of bacteria to polymorphonuclear leucocytes was 9 or 10 : 1, or in those shown on the right side, in which the multiplicity averaged 23: 1. By contrast, fluoride had a significant inhibitory effect on phagocytosis (p =
Energy for phagocytosis

17

Fig. 2. Phagocytosis of bacteria by leucocytes preincubated in buffer or iodoacetate. Apl )rox. x 400.(a) Normal leucocytes with intracellular staphylococci. (6, c) Leucocytes from the same experiment as (a), pretreatedwith 2 x 1O-pM iodoacetate, alsocontainingmanyintracellularstaphylococci.

these experiments were similar to those shown in fig. 1. After 45 min of phagocytosis, 42 % of the inoculum remained in the supernatant of flasks containing iodoacetate compared to 36 % of the inoculum in the flasks without inhibitor. This difference was not a significant one (p= > 0.5). The lack of inhibition of phagocytosis by iodoacetate was confirmed by examination of coverslip preparations of cells from the above experiments with staphylococci and streptococci. Similar numbers of intracellular cocci were regularly observed in normal and iodo2-

691802

acetate-treated cells. Representative cells are shown in fig. 2 a-c. Effect of iodoacetate on oxygen consumption and lactate production In order to confirm the inhibition of glycolysis by iodoacetate under the conditions of these experiments, respiration and lactate production were measured during phagocytosis of heatkilled staphylococci with and without iodoacetate. In fig. 3 is shown a typical experiment, in which complete inhibition of the respiratory kurst was observed in the presence of iodoExptl

Cell Res 56

18 Phyllis Bode1 & S. E. Malawista was present in flasks without inhibitor, compared to 6.6 % in flasks with iodoacetate. These results, then, seem to indicate that normal functioning of the glycolytic pathway in human blood polymorphonuclear leucocytes is not necessary for phagocytosis, under the conditions of these experiments.

Ordinate: ~1 Op. O---.-O, WBC+STAPH.; A--A, WBC+IAA+STAPH; A----A, WBC+F+STAPH; o-0, WBC. Fig. 3. Respiration of leucocytes (WBC) with and without iodoacetate (IAA) 2 x 1O-4 M and fluoride (F) 2 x 10V2 M during phagocytosis of heat-killed staphylbcocci (STAPH). Oxygen uptake of two flasks is averaged for each curve. Flasks contained 5 x 10’ leucocytes and 3.8 x lo* staphylococci. Bacteria were added at time zero (arrow).

acetate. Lactate production was estimated in the incubation supernatants in nine experiments, in which phagocytosis was allowed to proceed for 50 min after an initial leucocyte incubation of 40 min. Supernatants from flasks containing cells pre-treated with iodoacetate had an average lactate content of 280 ,ug/5 x 10’ cells, compared to supernatant from normal phagocyting cells which had an average value of 504 pug/5 X 10’ cells. These values are similar to those obtained by Sbarra & Karnovsky [23] in their study of phagocytosis by guinea pig exudate leucocytes, and suggest that there was a significant diminution of glycolysis after phagocytosis in the presence of iodoacetate. Others have reported about 70 ‘% suppression of lactate production in nonphagocyting human blood cells by 1 X 1O-4 M iodoacetate [22]. Experiments were also performed with iodoacetate in higher concentration, and without serum in the flask. Bacteria were opsonized by exposure to serum, centrifuged, suspended in buffer, and then added to flasks containing washed leucocytes in buffer. In four experiments, iodoacetate (5 x 1O-4 M) did not significantly suppress phagocytosis. Measurement of remaining supernatant bacteria after 40 min of phagocytosis showed that 3.8 % of the inoculum Exptl Cell Res 56

Effect of iodoacetate on granule lysis During examination of the thin coverslip preparations from some of the preceding experiments, we observed that leucocytes which had been pre-treated with iodoacetate before phagocytosis lacked the usual large, digestive vacuoles around the ingested bacteria (see fig. 2 a-c). Although many cells from flasks with iodoacetate showed some vacuolization, it was rare to find cells that were extensively vacuolated, whereas such cells were quite common in the control flasks. Similar observations were made on one-micron sections prepared from cell buttons fixed in glutaraldehyde (see Methods). As shown in fig. 4 a, b, the difference in size of vacuoles surrounding ingested bacteria is marked. These observations suggest that degranulation is impaired in the presence of iodoacetate. To confirm this impression, measurements of acid phosphatase activity were made in cellgranule and cell-supernatant fractions after phagocytosis. Previous studies have shown that most activity of this enzyme is found in the granule fraction before phagocytosis, but that after phagocytosis and degranulation only about one-third of the original activity remains associated with the granules [IO]. The results of a series of nine experiments are shown in fig. 5. There was a significant difference in the granuleassociated enzyme activity after phagocytosis when untreated cells were compared with cells pre-treated with iodoacetate (see fig. 5 b, c; p = ~0.01). In the presence of iodoacetate very little enzyme release occurred after phagocytosis. In fact, there was no significant difference (p = >0.05) between values for these cells and for normal non-phagocyting cells (see fig. 5 a, b). Cells incubated alone for 2 h with or without

Energy for phagocytosis

19

4. Phagocytosis of staphylococci by leucocytes preincubated in serum-buffer alone (a), with iodoacetate 2 x 1O-4 M (b), or with fluoride 2 x 1O-2 M (c). Onemicron sections, stained with metachromatic dyes. Approx. x 400. (a) Normal leucocytes with intracellular staphylococci. (b) Leucocytes pretreated with iodoacetate. Many intracellular bacteria are present but smaller vacuoles are present. (c) Leucocytes pretreated with fluoride. Cells are frequently swollen, vacuoles have appeared, some cells have ruptured, and few bacteria are inside the cells. Clumps of bacteria are seen in the medium. Fig.

iedoacetate showed the same granule-associated azyme activity (not shown). These results, then, i:dicate that degranulation after phagocytosis i impaired in the presence of iodoacetate. lffect of iodoacetate on killing c’ ingested bacteria

1). Although there was considerable variation among experiments in the per cent of inoculum recovered with the cells, the values with and without iodoacetate in each experiment tended of bacto be very similar. Thus, no inhibition terial killing at these time intervals was regularly observed in the presence of iodoacetate.

d some of the experiments reported in the prelive bacteria cding sections, cell-associated vere measured 20 and 40 min after phagocytosis i. cells with and without iodoacetate. There was n significant difference in the values (seetable

Effects of fluoride on phagocytosis and respiration Data presented above in fig. 1 indicate that sodium fluoride, 2 x 1O-2M, significantly inhibited Expti

Cell Res 56

20

Phyllis Bode1 & S. E. Mulawista

% of total acid phosphatase activity. 5. Effect of iodoacetate on loss of granule-associated acid phosphatase activity after phagocytosis. Average results + S.E.M. of nine experiments are shown. The difference between groups A and B is small (p = >0.05), whereas the difference between groups B and C is significant (p = ~0.01). A. WBC: B. WBC + IAA + STAPH: C. WBC + STAPH. Ordinate:

Fig.

phagocytosis. Intracellular bacteria were infrequently seen in cell sections (see fig. 4~). Similarly, no increased respiration was observed on addition of bacteria to fluoride-treated leucocytes (see fig. 3). Effects of fluoride on non-phagocyting cells

Fluoride has been reported to cause peculiar metabolic changes in guinea pig exudate granulocytes [23]. Increased oxygen uptake via the hexose monophosphate shunt occurs dramatically upon addition of this inhibitor to resting cells, and granule lysis has been reported [25]. When fluoride was added to human blood leucocytes, a small but consistent increase in respiration was observed for about 30 min. Since fluoride-treated cells exhibited extreme fragility, the morphology seen on thin coverslip preparations was unclear. Cell buttons were therefore fixed in glutaraldehyde, sectioned, and compared with cells incubated alone or with Table

1. Live cell-associated staphylococci per cent of inoculum

WBC WBC-IAA

20 mina

40 min

19.0+4.0+ (6)c 20.2 & 3.8 (6)

22.0 k 5.3 (6) 19.0+5.0 (6)

a Time of sample after bacteria added. b s.a. of mean. ’ Numbers in parentheses = number of experiments. Exptl

Cell Res 56

iodoacetate. Cells treated with fluoride appeared swollen, sparsely vacuolated, and were frequently ruptured, in contrast to normal cells or those incubated with iodoacetate. These effects of fluoride can be seen in fig. 4 c. Since these microscopic changes suggested alterations of membrane permeability, leucocyte preparations were incubated for 80 min, alone, with 2 x lo-* M iodoacetate or with 2 x 1O-2 M fluoride. Samples from each tube were taken before addition of inhibitors, and at the end of the incubation, for measurement of total leucocytes and eosin-positive cells. All initial samples contained 0 to 0.5 % eosin-positive cells. After incubation, 7 % of fluoride-treated cells were eosin-positive, compared to 1.5 % of control cells and 2 % of cells incubated with iodoacetate. Eight per cent fewer total white cells were counted after incubation in the fluoride-containing tube, compared to a change of 3 or 4% in count in the other tubes. These results suggest that fluoride, unlike iodoacetate, has profound effects on the morphological integrity of non-phagocyting cells. Because it is likely that these changes impair cell function, the inhibition of phagocytosis by fluoride is probably not due only to an inhibition of glycolytic activity. Changesin leucocyte adenosinetriphosphate (ATP) with phagocytosis

From the preceding data it seems apparent that phagocytosis of bacteria by polymorphonuclear leucocytes can occur without normal glycolysis. During phagocytosis rapidly available energy is probably required, however. Since ATP has been implicated in several biological systems as a source of energy for motility [19], and since a leucocyte-membrane ATP-ase has been described [28], it seemed of interest to measure leucocyte ATP levels before and after phagocytosis in our system. The results of these studies are shown in table 2. Levels of ATP did not change significantly during a 20-min incubation without bacteria, but were consistently lower after 20 min of phagocytosis. In four samples taken

21

Energy for phagocytosis

Table

2. Adenosine staphylococci

triphosphate

levels in leucocytes

before and after phagocytosis

of heat-killed

ATP mg/l x 108 WBC Cell source Human blood leucccytes Rabbit exudate leucocytes

Inhibitor

Before phagocytosis

IAA -

0.087f0.016a 0.080+0.015 0.057iO.009

(6)b (5) (8)

20 min after phagocytosis

20 min incubation (no phagocytosis)

0.056&0.012 (6) 0.018 kO.005 (5) 0.034+0.006 (8)

0.082~0.012c 0.038 iO.007 -

(3) (5)

a S.E. of mean. ’ Number in parentheses = number of experiments. ’ S.D.

10 min after addition of bacteria, a similar decrease was observed. Since phagocyting cells represent only about 60% of the total blood leucocyte population, the actual decrease of ATP in the phagocyting cells may be greater than that shown. In the presence of iodoacetate, levels of ATP decreased without phagocytosis, but reached even lower values after ingestion of bacteria. In order to confirm these studies with a population of cells consisting only of polymorphonuclear leucocytes, several experiments were performed with rabbit exudate cells (80 to 99 % polymorphonuclear leucocytes). These cells contained less measurable ATP, but a similar decrease in ATP was observed after phagocytosis. Coverslip preparations from sample flasks confirmed that active phagocytosis had occurred after 20 min incubation. These data suggest that ATP may play a role in phagocytosis. However, more evidence is clearly needed to implicate this or other highenergy compounds more directly with the cellular activities that accompany particle ingestion. DlSCUSSION Phagocytosis is a complex act involving a form of membrane and cytoplasmic activity that is still not clearly understood. It has been suggested [20] that the process of particle ingestion may be closely related to certain other cellular functions, such as pinocytosis of macromolecules, or secretion of cellular compounds, which also involve new membrane arrangements.

Therefore, studies of the energy requirements for phagocytosis may be of general physiological interest. In a number of previous studies [2, 9, 12, 23, 241, phagocytosis was modified by various inhibitors of glycolysis, including arsenite, iodoacetate and fluoride. On the other hand, compounds that interfere with Krebs cycle or cytochrome-linked oxidations, such as cyanide or dinitrophenol, appeared to be without effect. Even iodoacetate and fluoride, however, did not completely block phagocytosis in most studies. In the experiments reported here, iodoacetate had essentially no effect on phagocytosis, whereas fluoride reduced particle ingestion by at least 50 %. The difference between our results and those of previous workers may be due to several factors. In the first place, our studies were done with human blood leucocytes, whereas most previous work has employed exudate polymorphonuclear leucocytes, obtained from guinea pigs or rabbits. Besides possible differences in species, it is likely that differences exist between the metabolic and functional capacities of blood and exudate cells [5, 6, 151. However, in two experiments, rabbit exudate polymorphonuclear leucocytes appeared to phagocyte normally in our system in the presence of iodoacetate. Another difference between our studies and those of some others is in the multiplicity of particles to leucocytes employed. When ratios of bacteria to polymorphonuclear leucocytes did not exceed 25 : 1, no inhibition by iodoacetate was noted. At higher ratios, such as 50: 1, we Expil Cell Res 56

22 Phyllis Bode1 & S. E. Malawista have seen on coverslip smears some reduction in the numbers of bacteria per cell in the presence of iodoacetate, suggesting partially impaired function. If some critical factor is depleted successively with each act of phagocytosis, and if the new sources of energy supply are blocked, as is probably the case in the presence of iodoacetate, it might be expected that eventually an effect of the drug would become apparent. Since some previous studies [23, 241 have been done with very large multiplicities of particles to cells, a more pronounced effect may have been observed for this reason. Also, the size and nature of the particle used may alter the amount of energy required for ingestion. Polystyrene particles, sometimes of large size, have been used in several studies [9, 23, 241. Conditions of incubation, such as concentration of cells and particles and speed and intensity of ‘mixing, significantly alter the dynamics of phagocytosis [7], and may affect results with inhibitors. Arsenite has been shown to interfere with cell motility [8]. If iodoacetate has a similar action, it might appear to alter phagocytosis in some systems. The conditions of our experiments favor rapid phagocytosis, since frequent contacts between cells and bacteria occur. In our studies, iodoacetate completely suppressed the increased activity of the hexose monophosphate shunt following phagocytosis, as measured by increased oxygen uptake, and significantly impeded granule lysis, although it did not impair bacterial killing. A similar correlation between granule lysis and hexose monophosphate shunt activity has been observed in a number of different systems [16, 17, 21, 251. In our experiments fluoride significantly impaired phagocytosis, and caused swelling, vacuolization and rupture of cells. It is unlikely that the effect of this compound is due entirely to interference with glycolysis, since similar changes were not seen in cells incubated with iodoacetate at concentrations five times those reported to effectively block glycolysis. Since interference with glycolysis by iodoiodoacetate does not reduce phagocytosis in our experiments, it seems likely that preformed Exptl Cell Res 56

energy is available for this cellular function. Measurements of cellular ATP in the present study demonstrated that this compound was in fact rapidly depleted during phagocytosis, and more so in the presence of iodoacetate. Estimates from our data indicate a loss of about 1 x lo* molecules of ATP per ingested bacterial particle in the presence of iodoacetate. Woodin has shown that, after injury by leucocidin, extrusion of granule contents from leucocytes is greatly stimulated by added ATP [28]. He has suggested that ATP is an important compound in the maintenance of leucocyte membrane structure, in combination with calcium [27]. It seems likely that an early depletion of high energy compounds normally occurs during phagocytosis, and that increased glycolysis and other related metabolic activities then result in a restoration of high-energy levels and re-establishment of membrane integrity. P. Bode1 is research associate in medicine; S. E. Malawista is associate orofessor of medicine and senior investigator of the Arth;itis Foundation. The authors thank Dr Elisha Atkins and Dr J. W. Hollingsworth, in whose laboratories some of this work was done, for kind support, and Dr Klaus G. Bensch for providing the one-micron sections. We gratefully acknowledge the expert technical assistance of Gretchen V. Greene, Stella B. Cretella, and Ann Wechsler. This investigation was supported by grants from the United States Public Health Service (AI-01564, AM10493 and AI-271), and from the John A. Hartford Foundation. Part of this work was presented to the American Society for Clinical Investigation, Atlantic City, N. J., May 1968.

REFERENCES 1. Beck, W S, J biol them 232 (1958) 251. 2. Becker, H, Munder, G & Fischer, H, Hoppe-Seyler’s Z physiol Chem 313 (1958) 266. 3. Bergmeyer, H U (ed), Methods of enzymatic analysis, p. 266. Academic Press, New York (1965). 4. - Ibid p. 539. 5. Bode]. P T & Hollinesworth. J W. 3 clin invest 45 (1966j580. ’ 6. - Brit i exntl nathol49 (1968) I I. 7. Brandt:L, S&d j haematol (1967), Suppl. 2, 1. 8. Bryant, R E, DesPrez, R M, VanWay, M H & Rogers, D E, J exptl med 124 (1966) 483. 9. Cline, M J, Nature 212 (1966) 1431. 10. Cohn, Z A & Hirsch, J G, J exptl med 112 (1960) 1015. 11. Cohn, Z A & Morse, S E, J exptl med 1 IO (1959) 419. 12. - J exptl med 111 (1960) 667. 13. Elsbach, P, J clin invest 47 (1968) 2217. 14. Hanks, J H & Wallace, J H, Proc sot exptl biol 98 (1958) 188.

Energy for phagocytosis 15. Hartman, J D & Reidenberg, M, J appl physiol 12 (1958) 477. 16. Hirsch, J G & Cohn, Z A, J exptl med 112 (1960) 1005. 17. Holmes. B. Page. A R & Good. _R A., J clin invest 46 (1967) 1422. - ’ 18. Iyer, G Y N, Islam, D M F & Quastel, J H, Nature 192 (1961) 535. 19. Jahn, T L & Bovee, E C, Biochemistry and physiology of protozoa (ed S H Hutner) vol. 3, p. 62 (1964). 20. Karnovskv. M L. Phvsiol rev 42 (1962) 143. 21. Malawista,‘S E & Bddel, P T, J clin invest 46 (1967) 786. 22. Martin, S P, McKinney, G R & Green, A, Ann N Y acad sci 59 (1955) 996.

23

23. Sbarra, A J & Karnovsky, M L, J biol them 234 (1959) 1355. 24. Sbarra, A J & Shirley, W, J bacterial 86 (1963) 259. 25. Selvaraj, R J & Sbarra, A J, Nature 211 (1966) 1272. 26. Stahelin, H, Suter, E & Karnovsky, M L, J exptl med 104 (1956) 121. 27. Woodin, A M & Wieneke, A A, Biochem j 87 (1963) 487. 28. - Biochem j 90 (1964) 498.

Received August 23, 1968 Revised version received February 13, 1969

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