EFFECT OF OSMOLARITY ON PHAGOCYTOSIS

ANTHONY J. SBARRA, WILLIAM SHIRLEY,' AND JOHN S. BAUMSTARKh/epartment of Pathology and Medical Research, St. Margaret's Hospital, and Tufts University Medical

School, Boston, Massachusetts

Received for publication 20 August 1962

ABSTRACT

SBARRA, ANTHONY J. (St. Margaret's Hospital,Boston, Mass.), WILLIAM SHIRLEY, AND JOHN S.BAUMSTARK. Effect of osmolarity on phagocy-tosis. J. Bacteriol. 85:306-313. 1963.-The effectof a number of different compounds on phago-cytosis was studied. Phagocytosis was monitoredby morphological and biochemical means. It wasfound that the addition of compounds such asKCl, NaCl, sodium or potassium malonate, andK2SO4 to the phagocytic system inhibited phago-cytosis. The increased salt concentration specifi-cally inhibited the respiratory activity associatedwith phagocytosis. The endogenous respiration ofthe leukocytes was unaffected. Glycolysis and theincreased flow of glucose through the hexosemonophosphate pathway were also inhibited bythe elevated concentration of salts. In addition,cells exposed to high salt concentrations appearedto be reduced in size as compared with normalcells. The inhibition can be reversed by loweringthe salt concentration. It was suggested that theincreased osmotic pressure of the system was re-sponsible for the inhibition.

Malonate, a competitive inhibitor of succinicdehydrogenase, has been used as a tool for specifi-cally inhibiting the respiratory activity associatedwith the tricarboxylic acid cycle. This inhibitorhas also been used to study the nature of oxida-tion by leukocyte preparations. McKinney et al.(1953) indicated that the respiration arising fromhuman leukocytes is not derived from the tri-carboxylic acid cycle, since this respiration ismalonate-insensitive. In contrast, however,homogenates of leukocytes, which were exposedto malonate, showed a marked inhibition by thisagent. These findings thus suggested that therespiratory activity may in part arise from suc-

I Charlton Fellow, Department of Obstetricsand Gynecology, Tufts University School ofMedicine, Boston, Mass.

cinate, and that the insensitivity observed earlierwith whole cells was due to the inability ofmalonate to penetrate the cell membrane. Similarresults with whole cells (guinea pig and humanleukocytes) were obtained by Sbarra, Maney, andShirley (1961) and Sbarra, Shirley, and Bardawil(1962). However, these workers, in addition, wereable to demonstrate what appeared to be a directeffect of malonate on the increased respiratory ac-tivity which usually accompanies phagocytosis.These findings indicated that malonate couldenter the cell with the phagocytized particle.Direct evidence that such a phenomenon, referredto as "piggy back" phagocytosis (Sbarra et al.,1962), can occur was provided from studies withfluorescent-labeled rabbit -y-globulin. This fluo-rescent-labeled compound concentrates maxi-mally only in leukocytes that have ingested par-ticles. In the absence of particles, slight or nofluorescence is seen within the cell.The malonate inhibition of the respiratory ac-

tivity associated with phagocytosis was also ac-companied by an inhibition of particle uptake.Thus, malonate apparently was inhibiting thephagocytic process. Since phagocytosis has beenshown to be dependent on glycolytic energy(Sbarra and Karnovsky, 1959; Cohn and Morse,1960), it is difficult, in the light of the knownmechanism of inhibition by malonate, to conceiveof a mechanism by which malonate could inhibitthis process. Further studies with this inhibitorrevealed that an unusually high concentrationwas necessary for complete inhibition, and thatinhibition of respiration was not reversed by suc-cinate, oxaloacetate, malate, or fumarate. In fact,these tricarboxylic acid intermediates, used to re-verse the malonate inhibition, served as in-hibitors when tested alone. Since the observedresults could not be explained on the basis of thechemical nature of these intermediates, and sincethey were used as either the potassium or sodiumsalt, the role of these ions on phagocytosis wasexplored, and is the subject of the present report.

306

PHAGOCYTOSIS AFFECTED BY OSMOLARITY

MATERIALS AND METHODSThe general experimental approach was previ-

ously described in detail by Sbarra and Karnov-sky (1959, 1960) and Sbarra et al. (1962). Sus-pensions rich in polymorphonuclear leukocyteswere obtained from the peritoneal cavity ofguinea pigs (300 to 400 g) by the injection of a

12% solution of casein-sodium 16 to 18 hr beforethe experiment. The cells were then collectedfrom the peritoneum using a 0.9% sodium chloridesolution, centrifuged at 60 X g, and suspended inKrebs-Ringer medium without phosphate. Theamount of cellular material in the resulting sus-

pension was determined by measurement of totalcellular phosphorus (King, 1932). Phosphate was

then added to the suspension so that the leuko-cytes were finally suspended in the usual Krebs-Ringer phosphate medium (KRPM), buffered atpH 7.4.

Polystyrene latex particles, usually 1.171 ,u indiameter and suspended in buffered medium, were

used in all phagocytic experiments. The authorsare indebted to J. W. Vanderhoff and the DowChemical Co., Midland, Mich., for a generous

supply of these particles.All chemicals used were of analytical reagent

grade. Glucose-1-C'4 and glucose-6-C'4 were ob-tained from the New England Nuclear Corp.,Boston, Mass.The phagocytic experiments were carried out in

conventional Warburg manometric apparatus at37 C. The system consisted of polystyrene latexparticles, the leukocytic suspension, and the testcompound (made up in KRPM). After equilibra-tion, the experiment was started by adding theparticles and test compound from the side arm.

The ratio of polystyrene particles added with re-

spect to the leukocytes was approximately 100:1.When chemical analysis of the flask contents

was desired, the phagocytic process was termi-nated by immersion of a sample from the flasksto cold centrifuge tubes. The cells were thencentrifuged and washed once. The resulting super-natant fluid was collected, brought to volume,and analyzed. Lactic acid was determined by themethod of Barker and Summerson (1941).

Determination of the C14 distribution fromlabeled glucose was also performed in some experi-ments. The activity was determined after theincubation period by recovering the contents ofthe center well and converting it to BaC1403,which precipitated and could be counted. The

activities of glucose samples used as substratewere determined on their osazones. All radio-active measurements were carried out as previ-ously described (Sbarra and Karnovsky, 1959).

RESULTSThe effect of sodium or potassium malonate on

guinea pig polymorphonuclear leukocytes, at restand during phagocytosis, can be seen in Fig. 1.Neither compound showed any effect on the en-

dogenous respiratory activity of intact cells.Nevertheless, 300 Amoles of these agents com-

pletely inhibited the respiration associated withthe phagocytic process. Concentrations of lessthan 150,moles were without effect. The,umolesindicated (Fig. 1) were in addition to thosecontributed by the suspending medium. Themolarity of this suspending medium was

calculated to be 0.147 (441 ,umoles of salts);it was the same in all experiments. Thefinal pH in all cases was 7.4. This inhibi-tion, however, unlike the typical inhibitoryeffect of malonate, was not overcome by suc-

cinate, fumarate, malate, or oxaloacetate.These tricarboxylic acid intermediates were usedas sodium salts, potassium salts, or both, in con-

centrations ranging irom 30 to 300 ,umoles. Thedifference in 02 uptake due to phagocytosis equals100%; in the presence of 300 ,umoles of malonate,0%; in the presence of malonate plus each of

r Particlesun ParticlesIAbsent _ I Present

0

z

00 'so 30

,U MOLES Na ORt K+ MALONATE ADDED

FIG. 1. Effect of sodium and potassium malonateon phagocytosis. Guinea pig polymorphonuclearleukocytes were exposed to different concentrations ofeither sodium or potassium malonate, in the ab-sence and presence of polystyrene latex particles.Results are given on the basis of 100 jig of cellularphosphorus per 30 min. Resting cells (nonphagocytiz-ing) set at 100%. See text for additional details.

307VOL. 85, 1963

SBARRA, SHIRLEY, AND BAUMSTARK

TABLE 1. Effect of different concentrations of K+ on phagocytosisa

Amount of K Qo2 (phosphorus)" Particles per cellbJAmoles added6 Particles absent" Particles present 0 1-5 5-10 Over 10

[K+]0 8.5 i 0.6 33.4 4 2.0 15-17 1-6 3-8 71-81

pf < 0.CO175 7.4 i 2.0 35.8 4± 1.5 13-16 0-2 3-6 78-84

p <0.001150 8.9 i 0.8 29.3 4 1.6 12-28 1-5 4-6 63-83

p < 0.001300 8.2 :1 0.2 12.2 i 1.7 35-80 1-6 5-8 11-54

p > 0.05600 9.8 :1 1.0 12.1 :1 0.8 90-96 0.3 4-5 0-4

p > 0.1

a Results represent the average of four experiments.I Molarity of Krebs-Ringer Phosphate Buffer is 0.147 M. Its contribution to the final salt concentra-

tion is 441 ,umoles. Total volume of suspending medium, 3.0 ml.c Expressed as /Aliters of 02 uptake per 100lAg of cellular phosphorus per 30 min.d Results expressed as percentage of cells containing the designated number of particles.e Polystyrene latex particles 1.171,u in diam. See Fig. 1 for additional details.f The symbol p has the usual connotation as a probability value, referring to differences between

resting and phagocytizing cells. The mean and standard error of the mean is given in each case.

the intermediates mentioned above at differentconcentrations, 0%. In addition, it was previ-ously shown (Sbarra and Karnovsky, 1959) thatother respiratory inhibitors and uncoupling agentsdo not effect phagocytosis (KCN, dinitrophenol,and antimycin). Since the concentration ofmalonate required to achieve complete inhibi-tion of the increased respiratory activity ofthe phagocytizing cell was high with respect toeither K+ or Na+, the effect of these cations andother compounds on the increased respiratory ac-tivity associated with phagocytosis was investi-gated. Table 1 shows the effect of adding differentconcentrations of KCl on the increased oxygenuptake associated with particle entry. It can bereadily seen that 600 and 300 ,umoles of KCIsignificantly and selectively depress the respira-tory activity of phagocytizing cells, although therespiratory activity of nonphagocytizing cells(resting) remains substantially unaffected. As ex-pected, it can also be seen that the entry of par-ticles into the cell (followed by phase microscopy)is also inhibited and correlates with the inhibitedrespiratory activity.The effect of different concentrations of various

substances on phagocytosis is shown in Fig. 2. Allthe compounds examined at a concentration of300 ,moles, with the exception of glucose, signifi-

Giucoss

t ISO a00 450 Soo so oo 0oso00 00

,U MOLES ADDED

FIG. 2. Effect of adding increasing concentra-tions of different compounds on phagocytosis byguinea pig polymorphonuclear leukocytes. Seetext for additional details.

cantly inhibited the respiratory activity of leuko-cytes undergoing phagocytosis. The inhibitionwas relieved when the concentration was reducedto 75 ,umoles. The inhibition is also reversible.When leukocytes exposed to inhibitory concen-trations (300 MAmoles) of KCl for a 15-min periodare washed free of the salt, and are resuspendedin KRPM, they are equally as able to phago-cytize as are unexposed cells. (The difference in02 uptake due to phagocytosis equals 100%; inpresence of exposed unwashed cells, 0%; exposedwashed cells, 100%). The possibility that Na+was being pumped out of the cell in the presence

308 J. BACTERIOL.

PHAGOCYTOSIS AFFECTED BY OSMOLARITY

FIG. 3. Wet-mount preparation of guinea pig polymorphonuclear leukocytes incubated with polystyrenelatex particles in the presence of 600 ,umoles of KCI (left) and 0.6 ,moles of iodoacetate (right). 970 X. Seetext for additional details.

of added K+ or vice versa, and that this shift incation concentration inside the cell was effectingphagocytosis, was examined by simultaneouslyadding 150 ,umoles of NaCl and 150 ,umoles ofKCl to the phagocytic system. Phagocytosis wasinhibited to the same extent as when 300 ,umolesof KCl or NaCl were used, suggesting that theefflux of ions was not responsible for the observedeffect. Figure 3 shows the morphological appear-ance of the leukocytes and the particles after ex-posure to 600 ,umoles of KCl. The cells are rela-tively free from particles and there appear to beno particles on the periphery or near the cell. Themorphological appearance of leukocytes and par-ticles after exposure to iodoacetate is shown inFig. 3. This agent has also been shown to inhibitparticle entry (Sbarra and Karnovsky, 1959), andwas used for comparison. After exposure to iodo-acetate, the leukocytes are relatively freefrom particles; however, with this agent, in

contrast to the high salt-exposed cells, many par-ticles can easily be seen near the periphery of thecell. The control, showing the morphological ap-pearance of leukocytes and particles in the ab-sence of any added inhibitor, is shown in Fig. 4.The possibility that the inhibition was due to

the ionic strength of the solution was examined byadding increasing concentrations of KCl or NaCland K2SO4 to the phagocytic system. KRPMwas used as the suspending medium; its contribu-tion to ionic strength was 0.19. The final ionicstrength was varied by adding different concen-trations of KCl or K2SO4. The ionic strengthindicated is, therefore, due to the salt added. Inthe series with KCl or NaCl (Fig. 5), phagocytosiswas significantly inhibited at a concentration of300 ,umoles (0.1 M). This molarity gives an ionicstrength of 0.1. A given molarity of K2SO4 hasthree times the ionic strength of the same molarityof KCl or NaCl. Thus, 100 ,umoles (0.033 M) of

VOL. 85, 1963 309

SBARRA, SHIRLEY, AND BAUMSTARK

K2S04 has an ionic strength of 0.1, and, as canbe seen, does not have any inhibitory effect onphagocytosis. However, when the molarity ofK2SO4 is increased to 0.1 M (300 ,umoles; ionicstrength, 0.3), phagocytosis is completely in-hibited. These data indicate that the concentra-tion, and not the ionic strength, of a test com-pound is important in determining whether a cellwill or will not phagocytize.The KRPM used in these experiments had a

salt molarity of 0.147; this represents 441 ,umolesof salts added per reaction vessel. Phagocytosis isessentially unaffected until 150 ,umoles or more ofadditional salt are added. When 300 ,umoles ofsalt are added, phagocytosis is significantly in-hibited.The phagocytic process is accompanied by an

increased production of lactic acid and by an in-creased flow of glucose through the hexose mono-phosphate shunt (Sbarra and Karnovsky, 1959).

Xg:

cj40

o

b-

.05 0.10 0.15 0.20 0.25 0.30loNIc STat.NswN

FIG. 5. Effect of ionic strength on phagocytosis.See text for additional details.

0-

4

0"...-

a

z

be

FIG. 4. Wet-mount preparation of guinea pigpolymorphonuclear leukocytes incubated with poly-styrene latex particles. 970 X. See text for additionaldetails.

+ PAnrcLI

+ PAuTeCLI

ADDITIONS

FIG. 6. Effect of KCl and iodoacetate on lacticacid production. Results are based on the amountof lactic acid produced by 100lAg of cellular phos-phorus per 30 min. Resting (nonphagocytizing)cells set at 100%. See text for additional details.

Glycolytic inhibitors such as iodoacetate andsodium fluoride inhibit the formation of lacticacid and the increased flow of glucose through thehexose monophosphate pathway, as well as in-hibiting the phagocytic process. The effect ofsalts on these additional parameters was alsostudied. The increased production of lactic acidassociated with phagocytosis was inhibited by 600,umoles of KCl (Fig. 6). These results are similarto those obtained with 0.6 ,umoles of iodoacetate.The increased flow of glucose through the hexosemonophosphate pathway is also inhibited by 600j,moles of KCl (Table 2). The C-1:C-6 ratio risesapproximately fourfold as a result of phagocytosisbut, in the presence of either iodoacetate or KCl,the C-1: C-6 ratio is unaffected.

DISCUSSIONStudies on the effect of variations in osmotic

pressure and ionic strength on phagocytosis were

1 0411,K2S04KCINACI

310 J. BACTERIOL.

PHAGOCYTOSIS AFFECTED BY OSMOLARITY

TABLE 2. Effect of KCI and iodoacetate on C1402 production from glucose-Ji-C4 and glucose-6-Cl4a

Activity in C02

Additions C-1 C-6 C-1: C-6 PhagocytosisbParticles Particles Particles Particles Particles Particlesabsent present absent present absent present

None 916 10,654 32 100 29 106 NormalKCl (600 j,moles) 806 976 31 47 26 21 Minimallodoacetate (0.6 ,umoles) 541 1,311 65 144 8 9 Minimal

a Results are given on the basis of 100,ug of cellular phosphorus per 30 min. All counts were normalizedto 105 count/min added to the flasks as glucose.

I Normal indicates that over 80% of the cells have over ten particles per cell; minimal indicates thatover 80% of the cells have zero particles per cell. See Fig. 1 for additional details.

extensively reviewed by Mudd, McCutcheon, andLucke (1934) and Berry and Spies (1949). Perhapsthe most extensive work in this area is that ofHamburger (1912).A number of inorganic and organic compounds

were tested for their effect on phagocytosis; how-ever, the results obtained were inconclusive.Nevertheless, they did show that any of the com-pounds tested would, if used in a sufficiently highconcentration, disrupt the phagocytic process.The phagocytic process was judged by countingthe number of cells that had engulfed particles.

In an attempt to understand the mechanismsof phagocytosis, some unexpected findings wereencountered. Malonate, a compound whichstructurally resembles succinate and thereforecompetitively inhibits the formation of fumaratefrom succinate, was found to have an inhibitoryeffect on phagocytosis. Therefore, if the malonateinhibition is to be attributed to an inhibition ofsuccinic dehydrogenase, this inhibition should berelieved by increasing concentrations of succinateor by the addition of compounds, such as fu-marate, malate, or oxaloacetate, formed from suc-cinate. All of these agents were used in an effort toreverse the malonate inhibition. Not only didthese compounds fail to reverse the inhibition, butin addition they were found to be inhibitors ofthe phagocytic process. The inhibition by thesetricarboxylic acid cycle intermediates can now beexplained on the basis of a cation effect. This be-came evident when it was found that either KCIor NaCl at similar concentrations could mimic themalonate effect.The osmotic pressure of the test system has a

pronounced influence on the phagocytic capa-bility of the leukocytic cells. Phagocytosis occursmaximally in the presence of KRPM (0.147 M or

441 umoles). When 300 ,umoles of additional saltsare added to the phagocytic system, phagocytosisis significantly inhibited. This is true for all of theelectrolytes tested. However, in the case ofglucose, 600 ,umoles must be added before phago-cytosis is similarly affected. The inhibition ob-served with the nonelectrolyte is apparentlyidentical to that obtained with electrolytes, exceptthat in the former case approximately two tothree times as much compound must be used be-fore significant inhibition is achieved. Therefore,the mechanism responsible for the inhibitionnoted with the electrolyte and nonelectrolytemay be the same. This phenomenon is probablynot due to a utilization of the carbohydrate, as ithas been previously shown that only about 1% ofthe glucose added is used by the phagocytic cells.Further experiments, it is hoped, will elucidatethe nature of this phenomenon.The ionic strength of the test system is not an

important variable. This was evident when 100,umoles (0.033 M) of K2SO4, having an ionicstrength of 0.1, were without effect, and 300,umoles (0.1 M) of KCl, having an ionic strengthof 0.1, were quite effective in inhibiting phago-cytosis. It would thus appear that the electrolyticcharge brought about by these ions is withouteffect (in the concentrations of salts used in thissystem).The morphological appearance of the -cells after

exposure to KCl or NaCl and iodoacetate is ofinterest. It is obvious, in cells exposed to KCl orNaCl, that most of the cells are relatively freefrom particles, both inside the cell and along theperiphery of the cell. Further, the cells appear tobe smaller than the iodoacetate-exposed cells andthe control cells. In the iodoacetate-exposed cell,the intracellular localization of particles is again

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SBARRA, SHIRLEY, AND BAUMSTARK

relatively low, but one can clearly see that thereare many particles along the entire periphery ofthe cell. In the control cells, many particles canbe seen inside the cell as well as along its periph-ery. These morphological data indicate that theKCl- or NaCl-exposed cell may be unable to carryout phagocytosis, owing to a leakage of water andperhaps essential intermediates in the environ-ment. The cell in this state is thus incapable ofcarrying out some of the metabolic activitynecessary for phagocytosis. For example, glycoly-sis and the increased flow of glucose through thehexose monophosphate pathway are inhibited.The fact that the inhibitory effect of the addedsalts can be reversed supports this hypothesis.Further, these results also reveal that essentialmetabolites are probably not leaking out of thecell with the water, for if this were so, reversalwould not occur. It is conceivable, however, thatthe cell could resynthesize the essential metabo-lites. It is also possible that the particles are nolonger able to penetrate the "altered" cell mem-brane. The morphological appearance of the cellsexposed to 600 inmoles of KCl would tend to sup-port this line of reasoning.The possibility that the effect of either Na+ or

K+ was due to a release of one or the other cationfrom the cell was explored by adding both cationssimultaneously to the phagocytic system. Ourresults indicate that this is not the case. Theseobservations are in agreement with Elsbach andSchwartz (1959), who, using rabbit polymorpho-nuclear leukocytes, demonstrated that the trans-port of potassium in rabbit leukocytes occursindependently of sodium movements.

It is quite apparent from this work that the"malonate inhibition" observed with guinea pigleukocytes is actually a Na+ or K+ effect. In con-trast, the slight inhibition of the leukemic leuko-cytic respiration associated with phagocytosis by3 ,umoles of sodium malonate may be due to adirect effect of malonate and thus may be peculiarto the cell type (Sbarra et al., 1962). This ex-planation seems likely since similar concentra-tions of sodium malonate have no effect on therespiratory activity associated with phagocytosisof guinea pig leukocytes. Further studies havebeen undertaken to elucidate the malonate effectwith leukemic cells.

It is quite evident that the inhibition observedin these studies is specific. That is, only the

respiratory activity associated with phagocytosisis effected. The respiratory activity of resting cells(nonphagocytizing) is not altered. It is stronglyfelt that many of the conflicting reports cited inthe literature, regarding the effect of a number ofdifferent compounds on phagocytosis, may bedue to the effect of salts. It should be pointed outthat the effects of NaF and iodoacetate, effectivein low concentrations, cannot be explained as asalt effect, and appear to be due to a direct in-hibition of glycolysis.

ACKNOWLEDGMENTS

This work was supported by research grantC5307 from the National Cancer Institute and re-search grant G19531 from the National ScienceFoundation. The photography by George Daynesis greatly acknowledged, along with the typing ofthis manuscript by Doris Cote.

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BERRY, L. J., AND T. D. SPIES. 1949. Phagocy-tosis. Medicine 28:239-300.

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ELSBACH, P., AND I. L. SCHWARTZ. 1959. Studieson the sodium and potassium transport inrabbit polymorphonuclear leukocytes. J.Exptl. Med. 42:883-898.

HAMBURGER, H. J. 1912. Untersuchungen uberPhagozyten. J. F. Bergmann, Wiesbaden.

KING, E. J. 1932. The colorimetric determinationof phosphorus. Biochem. J. 26:292-297.

McKINNEY, G. R., S. P. MARTIN, R. W. RUNDLES,AND R. GREEN. 1953. Respiratory and gly-colytic activities of human leukocytes invitro. J. Appl. Physiol. 5:335-340.

MUDD, S., M. MCCUTCHEON, AND B. LUCKE. 1934.Phagocytosis. Physiol. Rev. 14:210-277.

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