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Carolina 27103. Received for publication 21 March 1974. Heat-killed ... human polymorphonuclear leukocytes than are the live organisms. The post- phagocytic ...
AND IMMUNITY, JUlY 1974, p. 25-29 Copyright © 1974 American Society for Microbiology

INFECTION

Vol. 10, No. 1 Printed in U.S.A.

Phagocytosis of Live Versus Heat-Killed Bacteria by Human Polymorphonuclear Leukocytes LAWRENCE R. DECHATELET, DEBRA MULLIKIN, PAMELA S. SHIRLEY, AND CHARLES E. McCALL Departments of Biochemistry and Medicine, The Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103 Received for publication 21 March 1974

Heat-killed Pseudomonas aeruginosa are phagocytized much more slowly by human polymorphonuclear leukocytes than are the live organisms. The postphagocytic increase in hexose monophosphate shunt activity (HMS) parallels the ingestion of the bacteria. The addition of serum to the live organisms causes a marked increase in both ingestion and cellular HMS activity; serum actually causes an inhibition of both uptake and HMS activity when added to the heat-killed organisms. Differences in postphagocytic HMS activity between live and heat-killed organisms were observed with three different species of bacteria, indicating that the phenomenon is not restricted to P. aeruginosa. These data emphasize that the influence of the particle on the phagocytic process is considerable. During the process of phagocytosis by human polymorphonuclear leukocytes, there are pronounced changes in cellular metabolism including increases in lactate production, oxygen consumption, hydrogen peroxide production, and oxidation of glucose via the hexose monophosphate shunt pathway (HMS) (6). The metabolic pathways of the cell have been investigated extensively, and several clinical conditions have been described in which these metabolic changes are deficient (1, 4). Various laboratories have employed a wide range of particles including latex, zymosan, and various strains of heat-inactivated bacteria to induce phagocytosis. Relatively little attention has been paid to the influence of the type of particle employed on particle-cell interaction. In the present communication we suggest that the nature of the particle may exert a considerable influence on postphagocytic metabolism. We demonstrate here that live and heat-killed bacteria may interact differently with cells, as judged by uptake of radiolabeled bacteria or by stimulation of cellular HMS activity.

Corp., Buffalo, N.Y.) was added to each. The tubes were mixed by inversion, and the red blood cells were allowed to sediment at room temperature for 20 min. All subsequent steps were performed at 4 C. The white cell-rich plasma was drawn off and centrifuged at 500 rpm for 8 min. The supernatant solution, containing most of the platelets, was discarded, and the white cells were washed once with 0.9% saline. The washed white cells were suspended in 6 ml of cold deionized water to hemolyze contaminating red cells. After 10 s, 2 ml of 3.5% sodium chloride was added to restore isotonicity. After centrifugation, the cells were suspended in phosphate-buffered saline (PBS), counted by conventional means, and adjusted to a final concentration of 5 x 106 phagocytes per ml by the addition of PBS. Phagocytes were defined as segmented neutrophils, band neutrophils, eosinophils, and monocytes. Viability was determined by the ability of the cells to exclude 1% trypan blue dye. Preparation of particle suspensions. Stock cultures of bacteria were incubated overnight in Trypticase soy broth to the stationary phase of growth. When heat-killed bacteria were used, the flask was immersed in a boiling-water bath for 20 min. For experiments comparing live and heat-killed bacteria, a single culture was divided into two parts and only one of these was subjected to the boiling-water bath. The bacteria were collected by centrifugation, washed three times with 0.9% sodium chloride, and suspended in PBS to a standard absorbance of 0.20 at 525 nm on a Beckman DU spectrophometer. All bacterial suspensions were sonically treated for 15 s prior to use (Branson sonic oscillator with a microtip and power output of approximately 20 W) to ensure a uniform distribution of particles. Control experiments demon-

MATERIALS AND METHODS Isolation of cells. Approximately 50 ml of blood was collected into a syringe containing approximately 500 U of heparin. Donors were apparently healthy volunteer subjects. The heparinized blood was divided into 8-ml volumes, and 2 ml of plasma gel (HTI 25

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strated that this degree of sonic treatment did not affect the viability of live bacteria; examination of the sonically treated suspension by phase microscopy indicated good dispersion with a minimum of bacterial clumping. Glucose oxidation. The oxidation of [1- "C]glucose to "CO2 was determined by modifications of a previously described method (2). Each flask contained in a total volume of 3.0 ml: 0.20 tCi of [1-'4C]glucose (specific activity 54.2 mCi/mmol), 0.30 ml of normal human serum, 1.0 umol of KCN, and 1.0 ml of the appropriate particle suspension in PBS. Nonphagocytizing flasks received 1.0 ml of PBS in place of the particle suspension. The KCN is added to ensure that the glucose oxidation measured is due to the CN-insensitive respiratory burst; it is not employed in the phagocytic assay. The concentration of CN- used has no effect on phagocytosis per se and does not affect the viability of the live organisms (unpublished observations). Reaction was initiated by the addition of 1.0 ml of isolated human phagocytes (5 x 10' cells). The final volume was adjusted to 3.0 ml with PBS. In those experiments in which the serum concentration was varied, the glucose concentration of the serum was assayed by the glucose oxidase procedure (5), and appropriate quantities of glucose solution were substituted for the serum so that the final concentration of glucose was the same in all flasks. This was necessary to avoid changes in the specific activity of the [1- "C ]glucose in the various experimental flasks. Reaction was allowed to proceed for 1 h at 37 C and was terminated by the addition of 1.0 ml of 5% trichloroacetic acid. '4C02 released during the course of the incubation was collected in 0.50 ml of hyamine hydroxide and counted in a liquid scintillation spectrometer as previously described (2). Phagocytosis assay. Phagocytosis of bacteria was quantitated by measuring leukocytic uptake of radiolabeled organisms. Pseudomonas aeruginosa was incubated overnight at 37 C in 10 ml of Trypticase soy broth containing 100 qCi of uniformly "C-labeled protein hydrolysate (New England Nuclear Corp., Boston, Mass.). The bacterial suspension was divided into two parts on the following day, and one portion was heat-killed by immersion in a boiling-water bath for 20 min. Both the live and heat-killed bacteria were washed three times with cold 0.9% saline and diluted to the standard absorbance of 0.20 at 525 nm. For the phagocytic assay, leukocytes were suspended at 5 x 106 cells/ml in PBS. A 1.0-ml amount of leukocyte suspension was incubated with an equal volume of labeled bacteria and varying amounts of type 0 serum in a total volume of 3.0 ml. The serum was collected from at least five separate donors, pooled, and stored in small volumes at -70 C. Each volume was thawed only once prior to use; a fresh pool of serum was prepared every 30 days. The reaction was stopped after varying periods of time by the addition of 4.0 ml of cold 0.04 M sodium fluoride in PBS. The cells were collected by centrifugation at 800 x g for 10 min, and the pellets were washed free of noningested bacteria by repeated suspension in 5.0 ml of 0.01 M sodium fluoride in PBS containing 10%

fetal calf serum. After three such washes, the pcilet was dried overnight in a 60 C water bath, and digestcd in 0.50 ml of 0.2 N NaOH for 4 h. The solutions were then neutralized with 0.20 ml of 3% glacial acetic acid, 0.50 ml of deionized water was added, and 1.0-ml volumes were counted in 10 ml of Aquasol (New England Nuclear Corp., Boston, Mass.) with a liquid scintillation spectrometer.

RESULTS Figure 1 shows the uptake of live and heatkilled P. aeruginosa as a function of time. Each incubation flask contained serum in a final concentration of 10%. The live bacteria are ingested very readily; the heat-killed organisms are taken up very slowly under the conditions employed. The stimulation of HMS activity (Fig. 2) parallels the data on ingestion of the bacteria. The lag period observed at the early time periods simply reflects the fact that HMS stimulation follows the ingestion process in a time sequence; this has been described previously (3). At all time points examined, the live bacteria stimulate the HMS of polymorphonuclear leukocytes to a far greater extent than does a similar number of heat-killed organisms. Figure 3 illustrates the effect of varying serum concentration on the phagocytosis of P. aeruginosa. Incubation time in this experiment

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FIG. 1. Ingestion of radiolabeled Pseudomonas aeruginosa by human polymorphonuclear leukocytes. Solid line. Heat-killed bacteria; dotted line, live bacteria. The data are taken from a single experiment which is representative of three separate experiments. Each point was determined in duplicate in each experiment.

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VOL. 10, 1974

was 30 min. The ingestion of live bacteria is markedly stimulated (threefold) by the addiof serum; maximal stimulation is achieved tion 15/0, with 0.10 ml of serum per incubation flask. In the case of both the live and heat-killed bacteria, there is significant ingestion in the absence of serum. The addition of 0.05 ml of serum to the heat-killed bacteria, however, results in 50% inhibition of the ingestion. Increasing quantities -I0 of serum result in a slightly greater degree of 1::, /~~~~~ ingestion, but this does not return to the level O/ seen in the absence of serum. Figure 4 illustrates the parallel study measuring HMS activity. Again, the stimulation of 5 HMS activity by live bacteria is markedly enhanced by the presence of serum. The stimulation of HMS activity by the heat-killed organisms, on the other hand, is inhibited by the addition of a small amount (0.05 ml) of serum. II / The general features of the serum effect of HMS 15 20 10 5 stimulation parallel those observed with the Time (min) phagocytosis assay, indicating that the stimulaFIG. 2. Effect of Pseudomonas aeruginosa on the tion of the HMS in this system is a fairly oxidation of [1-14Clglucose by human polymorphonu- accurate reflection of the extent of phagocytoclear leukocytes. Solid line, Heat-killed bacteria; S's. dotted line, live bacteria. Each point represents the That this phenomenon is not peculiar to P. average of triplicate determinations. All values have aeruginosa is illustrated by the results in Fig. 5. been corrected for the metabolism of the bacteria The effect of serum concentration on the HMS alone. stimulation by three types of bacteria is seen. In all cases, the maximal stimulation in the presence of serum is greater with live than with heat-killed bacteria, although there are signifi0 cant differences among the various bacteria. 1' The degree of [1-14C]glucose oxidation obtained 301in the absence of serum for the heat-killed and (J

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FIG. 3. Effect of serum concentration on the ingestion of radiolabeled Pseudomonas aeruginosa by human polymorphonuclear leukocytes. Solid line, Heat-killed bacteria; dotted line, live bacteria. The data are taken from a single experiment which is representative of three separate experiments. Each point was determined in duplicate in each experi-

FIG. 4. Effect of serum concentration on [114C]glucose oxidation of human polymorphonuclear leukocytes induced by live and heat-killed Pseudomonas aeruginosa. Solid line, Heat-killed bacteria; dotted line, live bacteria. Each point represents the average of triplicate determinations. All values have been corrected for the metabolism of the bacteria

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not nearly as great as the ingestion of the live organism. This difference is reflected in the 5.0 h r postphagocytic stimulation of HMS activity, stimulation the degree of the of measure albeit ofindirect, accurate, HMS is an that ,*--- indicating mQ 30 &0 the extent of' ingestion. In addition to the 3.0 in degree of phagocytosis between the difference 20+' r 40 2.0'2.0 4 live and heat-killed organisms, there is also a ~ ~ 2o 102marked difference in the serum requirement for 20^; 10O phagocytosis. Ingestion of' the live bacteria is 20 40 markedly enhanced by the addition of a small 20 40 20 40 ml Serum amount of serum; ingestion of the heat-killed FIG. 5. Effect of serum concentration on [1- bacteria is actually inhibited by the same 14C]glucose oxidation of human polymorpnonuclear leukocytes induced by three species of live and amount of serum. This phenomenon is not heat-killed bacteria. Solid line, Heat-killed bacteria; specific to P. aeruginosa as indicated by the dotted line, live bacteria. Each point represents the data in Fig. 5; three dif'ferent bacteria show average of triplicate determinations. All values have differences in the postphagocytic HMS activity been corrected for the metabolism of the bacteria depending upon whether or not they have been heat-inactivated. Although there are marked alone. differences in glucose metabolism between the live and heat-killed bacteria, the inhibition by amounts of' serum with the killed orgalivaebacteia hv been normalized to the same small value in this experiment to simplia. compari- nism is not always evident. The reasons for these differences are not sons among the different bacteria. clear. Differences in methodology do not supply DISCUSSION a reasonable explanation, since equal numbers Several recent studies have emphasized the of live and heat-killed organisms were employed fact that the metabolic response of the leuko- in parallel experiments (as determined by the cyte after phagocytosis is at least partially a absorbance of the bacterial suspensions at 525 function of the nature of the ingested particle. nm and confirmed by the direct count of' the Krenis and Strauss (7) and Roberts and Quastel suspensions in a Petroff-Hauser counter). The (10) investigated the effect of particle size, and amount of radioactive label in the two preparademonstrated that the postphagocytic oxygen tions was similar as determined by direct scinconsumption was proportional to the size and tillation counting of an volume of each bacterial quantity of the latex particles ingested. Man- suspension. Finally, each suspension was briefly dell (9) extended this observation by demon- sonically treated before use to ensure a unif'orm strating that phagocytosis of heat-killed bacte- distribution of' particles; microscope examinaria resulted in a greater increase in oxygen tion indicated no gross differences in the degree consumption and hydrogen peroxide production of clumping between the live and the heatthan did the phagocytosis of latex particles, killed organisms. It is possible that the heating causes changes even though the particle size was comparable. He made no attempt to relate the magnitude of' in the surface of the bacteria which render them the metabolic change to the degree of ingestion, less suceptible to phagocytosis. One possibility however. Lehrer (8) investigated the effects of might be that specific binding sites for serum colchicine and chloramphenicol on the oxida- factors are changed so that the bacteria cannot tive metabolism of' human neutrophils as a be opsonized normally, resulting in a decrease function of the type of particle used. He re- in phagocytosis. The dif'ferential effect of serum ported quite different effects of heat-killed on the uptake of the live and heat-killed bacteStaphylococcus aureus, heat-killed Candida al- ria is certainly compatible with such an explabicans, and latex particles, and correctly em- nation. An alternate possibility is that the neutrophil phasized that the effect of drugs on metabolism of neutrophils challenged with one particle can somehow distinguish between viable and should not be extrapolated to predict their nonviable organisms and preferentially ingest performance with another. The present results only the virulent ones. Teleologically, it would support these observations and demonstrate be very useful to an organism if' it possessed this that the nature of the particle exerts a more capability. Whatever the explanation, these data suggest subtle influence than was previously suspected. The ingestion of heat-killed P. aeruginosa is that results obtained with heat-killed bacteria S albus

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may not entirely reflect the situation with living bacteria. These observations emphasize the complexity of the phagocytic process and indicate that the nature of the particle used to challenge the cells must be carefully controlled.

ACKNOWLEDGMENTS This research was supported by a grant from the Forsyth Cancer Service, Public Health Service grant AI-10732 from the National Institute of Allergy and Infectious Diseases, and Public Health Service grant CA-12197 from the National Cancer Institute. LITERATURE CITED 1. Cooper, M. R., L. R. DeChatelet, C. E. McCall, M. F. Lavia, C. L. Spurr, and R. L. Baehner. 1972. Complete deficiency of leukocyte glucose-6-phosphate dehydrogenase with defective bactericidal activity. J. Clin. Invest. 51:769-778. 2. DeChatelet, L. R., M. R. Cooper, and C. E. McCall. 1971. Dissociation by colchicine of the hexose monophosphate shunt activation from the bactericidal activity of the leukocyte. Infect. Immunity 3:66-72. 3. DeChatelet, L. R., P. Wang, and C. E. McCall. 1972.

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Hexose monophosphate shunt activity and oxygen consumption during phagocytosis: temporal sequence. Proc. Soc. Exp. Biol. Med. 140:1434-1436. Holmes, B., A. R. Page, and R. A. Good. 1967. Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J. Clin. Invest. 46:1422-1432. Kaplan, N. 0. 1957. Enzymatic determination of free sugars, p. 107-110. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3, Academic Press Inc., New York. Karnovsky, M. L. 1968. The metabolism of leukocytes. Seminars Hematol. 5:156-165. Krenis, L. J., and B. Strauss. 1961. Effect of size and concentration of latex particles on respiration of human blood leukocytes. Proc. Soc. Exp. Biol. Med. 107:748-750. Lehrer, R. I. 1973. Effects of colchicine and chloramphenicol on the oxidative metabolism and phagocytic activity of human neutrophils. J. Infect. Dis. 127:40-48. Mandell, G. L. 1971. Influence of type of ingested particle on human leukocyte metabolism. Proc. Soc. Exp. Biol. Med. 137:1228-1230. Roberts, J., and J. H. Quastel. 1963. Particle uptake by polymorphonuclear leukocytes and Ehrlich ascites-carcinoma cells. Biochem. J. 89:150-156.