Effect of Phagocytosis by Human Polymorphonuclear Leukocytes and Rabbit Alveolar Macrophages on 2-deoxyglucose Transport MIN-FU TSAN Divisions of Hemafotogy and Nuclear Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205
ABSTRACT 2-Deoxyglucose transport was characterized in human polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (AM). The Km was 1 mM for human PMN and 1.6 mM for rabbit AM, and the Vmax was pmoles/45 sec/106 AM. pmoles/45 sec/106 PMN and 5.09 x 0.66 x The rate of 2-deoxyglucose transport was the same before and after phagocytosis in PMN from normal individuals and three patients with chronic granulomatous disease, as well as rabbit AM. Studies of the kinetics of 2-deoxyglucose transport and intracellular fate of 2-deoxyglucose in human PMN indicate that the nature of the membrane transport system is not altered by phagocytosis. The results support the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites. Phagocytosis and carrier-mediated membrane transport are two distinct membrane functions by which substances are translocated across the plasma membrane. During phagocytosis, a portion of the plasma membrane is internalized (Essner, '60; Korn and Weisman, '67; Tsan and Berlin, '71a). We (Tsan and Berlin, '71a) have previously demonstrated that in five separate transport systems in rabbit polymorphonuclear leukocytes (PMN) (adenosine and two adenine transport systems) and alveolar macrophages (AM) (adenosine and lysine transport systems), the rate of transport was unaffected even after a large portion of the membrane had been internalized. We (Tsan and Berlin, '71a) provided evidence that transport sites (carriers) were preserved on the cell surface during internalization of the plasma membrane which accompanied phagocytosis. The results are consistent with the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites. Confirmatory observations have recently been reported by others (Straus et al., '77a,b). Phagocytosis is accompanied by a marked stimulation of glucose oxidation, especially through the hexose monophosphate shunt (Sbarra and Karnovsky, '59). This increased glucose utilization can be accomplished by J. CELL. PHYSIOL. (1979) 99: 23-30.
accelerated glycogen breakdown (glycogenolysis) or derived from extracellular source (Sbarra and Karnovsky, '59; Stossel e t al., '70). Therefore, I extend our previous observation to study the effect of phagocytosis on glucose transport utilizing 2-deoxy-D-glucose, a glucose analog. The results of this study form the basis of this report. MATERIALS AND METHODS
Chemicals I4C-D-glucose, 14C-2-deoxy-D-glucose and 14C-lysine (all uniformly labelled) were obtained from New England Nuclear, Boston, Massachusetts. Ficoll was obtained from Pharmacia Fine Chemicals, Inc., Uppsala, Sweden and sodium diatriazoate (Hypaque) from Winthrop Laboratories, New York. Polystyrene latex (0.79 pm in diameter) was obtained from the Dow Chemical Co., Indianapolis, Indiana. The preparation of leukocytes and alveolar macrophages Polymorphonuclear leukocytes were isolated from normal individuals and three patients with x-linked chronic granulomatous Received Oct. 12, '18.Accepted Nov. 16, '78. I Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; AM, alveolar macrophages; CGD, chronic panulomatous disease.
23
24
MIN-FU TSAN
disease (CGD) as described previously (Tsan et al., '76). Briefly, venous blood was obtained and leukocytes were isolated by dextran sedimentation of red blood cells, differential centrifugation and NH,Cl lysis of contaminating red cells. The leukocytes then were suspended in 10 ml of modified Hanks' solution (Tsan and Berlin, '71b) with 5 mM glucose, and placed on top of a 10-ml Ficoll-Hypaque mixture and centrifuged a t 400 g for 40 minutes a t 20°C according to Boyum ('68). The pellet which consisted of 97-99%pure PMN by differential counting was washed twice with modified Hanks' solution (5 mM glucose). Rabbit alveolar macrophages were obtained by the method of Myrvik et al. ('61) as modified by Tsan and Berlin ('71b). Cell monolayers used in all experiments were formed as previously described on round glass cover slips at a cell density of 2 million PMN and 0.3 million AM, per cover slip (Tsan and Berlin, '71a,b). Determination of membrane transport by a rapid-sampling technique The technique developed by Hawkins and Berlin ('69) was used to characterize the glucose transport system and study the effect of phagocytosis on glucose transport in human PMN and rabbit AM. After the formation of cell monolayers, the coverslips were pre-incubated with modified Hanks' solution without glucose for 20 minutes to deplete intracellular and remove extracellular glucose. The uptake of radioactive glucose in 45 seconds, which represents the initial rate of transport before and after phagocytosis, then was determined. This value was corrected for diffusion and extracellular contamination in order to obtain the carrier-mediated transport. The radioactivity recovered a f t e r incubation of monolayers a t 37°C a t supersaturating concentration of substrate (40 mM glucose) was considered to be the sum of diffusion and contamination components (Tsan and Berlin, '71b). In order to determine whether glucose and 2-deoxyglucose share the same transport system, competitive studies were carried out measuring the uptake of 0.1 mM ''CC-glucose or 0.1 mM 14C-2-deoxyglucosein the presence of 20 mM glucose or 2-deoxyglucose. These studies showed t h a t 2-deoxyglucose and glucose shared the same transport system, and subsequent studies were performed with 2-deoxyglucose only. The intracellular fate of 2-deoxyglucose was analyzed by ascending paper
chromatography with two solvent systems (tbutanol : methylethylketone : formic acid : H 2 0 , 40:30:15:15, and n-butanol : ethanol : HzO, 52:33: 15) as described previously (Tsan and Berlin, '71b). For the determination of membrane transport after phagocytosis, monolayers were first incubated with media containing 1%by weight of polystyrene latex particles (0.79 p in diameter) and 2 mM Mg++ in modified Hanks' solution without glucose for 20 minutes a t 37°C. Maximal phagocytosis was achieved under this experimental condition. At the end of the incubation, the coverslips were drained and washed with modified Hanks' solution at room temperature, then the rate of 2-deoxyglucose transport was determined. Controls were done with the media containing no particles and the rate of transport determined in the same way (Tsan and Berlin, '71a). All experiments were done with duplicate monolayers and the results averaged. The results are expressed as the amount of 2-deoxyglucose transported per 45 seconds per monolayer. RESULTS
The kinetics of 2-deoxyglucose transport in human PMNand rabbitAM Initial rates of 2-deoxyglucose were measured a t substrate concentrations between 0.1 mM and 4 mM. The radioactivity per coverslip was corrected for diffusion and extracellular contamination (Tsan and Berlin, '71b), and converted to wmoles of 2-deoxyglucose transported per monolayer. The data were expressed in a double reciprocal plot, from which the Km and Vmax were calculated. The derived values of Km and Vmax of 2-deoxyglucose in both cell types were quite constant. The results of typical experiments are shown in figure 1 for human PMN and figure 2 for rabbit AM. The Km was 1mM and the Vmax pmoles/45 sec/106cells was about 0.66 x for human PMN. The Km and Vmax for rabbit AM were 1.6 mM and 5.09 X lo-' pmoles/45 sec/106 cells respectively. These Km values are similar to those obtained in guinea pig peritoneal exudate PMN and macrophages (Straus et al., '77a,b; Bonventre and Mukkada, '74). Effect ofphagocytosis on 2-deoxyglucose transport The rate of 2-deoxyglucose transport was measured a t 1 mM substrate concentration
25
PHAGOCYTOSIS AND 2-DEOXYGLUCOSE TRANSPORT
l/C*"C.(tnM) Fig. 1 Kinetics of 2-deoxyglucose transport in human polymorphonuclear leukocytes. Cell monolayers were incubated in the presence of 'C-2-deoxyglucose in concentrations from 0.1 mM to 2 mM for 45 seconds. pmolesI45 sec/2 million cells. The initial rate (V)was expressed as
60
50
40
.>
\
-1
30
0
1
2 3 I/Conc. (mM)
4
5
Fig. 2 Kinetics of 2-deoxyglucose transport in rabbit alveolar macrophages. Cell monolayers were incubated in the presence of "C-2-deoxyglucose in concentrations from 0.2 mM to 4 mM for 45 seconds. The initial pmoles/45 sed0.3 million cells. rate (V)was expressed as
26
MIN-FU TSAN TABLE 1
Effect of phagocytosis on 2-deoxyglucose transport by human polymorphonuclear leukocytes and rabbit alveolar macrophages Rate of transport ' (?SE)
Polymorphonuclearleukocytes Alveolar macrophages
Control
Phagocytosis
7.0420.71(7)3 0.5320.04(7)
6.3520.82(7) 0.54-tO.07(7)
P values
> 0.40 >0.70
' The rate of transport was determined at 1 mM 2-deoxyglucose and expressed as (lo-' ,~molesJ45sec per 2 million cells) for p l y morphonuclear leukocytes and (10.' pmolesJ45 Bec per 0.3 million cells) for alveolar macrophages. P values were determined based on pair differences. Numbers in parentheses = number of experiments, each experiment was done with duplicate monolayers and results averaged.
after the cells were induced to phagocytize polystyrene latex particles for 20 minutes. The results are shown in table 1. It is obvious that the rate of 2-deoxyglucose transport was not significantly different after phagocytosis. The kinetics of carrier-mediated membrane transport follows the enzyme kinetics and the initial reaction is often represented by the following model (Tsan and Berlin, '71a), ki kz (a) C + S e s C S - C + S i k3
where C represents carrier, and Se and Si represent extracellular and intracellular substrate concentrations, respectively. CS represents the concentration of the carrier-substrate complex. The k's (kl, k 2 and k,) represent the rate constants. If Ct represents the total number of carriers, then the maximal transport rate can be expressed as: (b) Vmax
=
kz [CtSI.
Thus, Vmax is proportional to the total number of carriers under each experimental condition. The initial rate (V) of transport at a single concentration S is determined by the following equation:
From equation (c), i t is apparent that three factors can influence the initial rate, namely, k,, [CtSI, and Km. The number of carriers are reflected by the initial rates under various experimental conditions only when k 2 and km remain constant. Therefore, i t is of prime importance to find out whether these factors are changed by phagocytosis. It is assumed that in these experiments k, can be affected only by exchange diffusion (Tsan and Berlin, '71a).
Effect of phagocytosis on the kinetics of 2-deoxyglucose transport Kinetic studies of 2-deoxyglucose transport in human PMN with and without phagocytosis were carried out and the results as expressed by double reciprocal plot are shown in figure 3. The Km and Vmax remained the same after phagocytosis. Therefore the rates of transport shown in table 1, as determined a t a single substrate concentration, are satisfactory indices of the Vmax of the 2-deoxyglucose transport system. Effect of preloading with glucose on 2-deoxyglucose transport The stimulation of tracer influx by high intracellular substrate concentrations has been repeatedly described and designated exchange diffusion (Heinz and Walsh, '58; Tsan and Berlin, '71b). This is presumably because of an increase in k, in equation (c) (Tsan and Berlin, '71a). I t is possible that the intracellular concentration of glucose increased during phagocytosis due to enhanced glycogenolysis (Stossel et al., '70). Although the affinity of 2deoxyglucose for the carrier (Km) remains unchanged, the number of carrier may be reduced but compensated by an increased rate of transport because of exchange diffusion (i.e., k, increased) secondary to increased intracellular glucose concentration after phagocytosis. In order to rule out this possibility, human PMN were preloaded with 10 mM glucose for one to ten minutes before the rate of 2-deoxyglucose transport was determined. Preloading with 10 mM 2-deoxyglucose resulted in cell damage and PMN detached from the coverslips. If one assumes that the transported glucose is not metabolized, preloading with 10 mM glucose for ten minutes results in
PHAGOCYTOSIS AND 2-DEOXYGLUCOSE TRANSPORT
-
Control Phagocytosis
l/Conc.(rnM) Fig. 3 Kinetics of 2-deoxyglucose transport in human polymorphonuclear leukocytes with and without phagocytoais. Cell monolayers were first incubated with 1% polystyrene latex in the absence of glucose for 20 minutes, rinsed, and then 45-second uptake of “C-2-deoxyglucose in concentrations from 0.2 mM to 4 mM was measured. Control was incubated in the absence of particles. V was expressed as ~ m o l e s / 4 5secA million cells.
10
8
......w . * ............ )
c L
o
0
e..........................................
8””””’........
e
0
a 2 6
E
I
0 0)
4 0
c
a
a
Control
e Phagocytosis
2
Fig. 4 Effect of glucose-preloading on 2-deoxyglucoae transport by human polymorphonuclear leukocytes with and without phagocytosis. Cell monolayers were first incubated with 1%polystyrene latex, rinsed and then incubated with 10 m M glucoae for one to ten minutes. Cell monolayers were rinsed and the rate of 2-deoxyglucose transport was then determined. Control was incubated in the absence of particles. The rate of 2deoxyglucose transport was expressed a s pmoles/45 s e c h million cells.
27
28
MIN-FU TSAN TABLE 2
Effect ofphagocytosis on 2-deoxyglucose and lysine transport by polymorphonuclear leukocytes from three patients with chronicgranulomatous disease Rate of transport (&SE)
2-deoxyglucose Lysine
Concentration
Control
Phagocytosis
P value
1 mM 0.1mM
6.49k2.12(5)3 1.5650.34(3)
6.3951.41(5) 1.57-tO.25(3)
>0.80
' The rate of transport was expressed as (10.'
im1olesI45 sec per 2 million cells) for 2-deoxyglucose and
>0.60 pmolesl45 see per 2
million cells) for lysine. P values were determined based on pair differences. Numbers in parentheses = number of experiments. Each experiment was done in duplicate and results averaged.
'
an intracellular concentration of glucose of 2.5 mM (assuming PMN cell water of 0.346 pl/million cells) (Hawkins and Berlin, '69). However, this is not true since most of t h e transported glucose is metabolized (see below). As shown in figure 4, preloading with glucose did not affect the rate of P-deoxyglucose transport by human PMN with and without phagocytosis. Thus, there was no exchange diffusion phenomenon under these experimental conditions.
Intracellular fate of transported 2-deoxyglucose The fate of 2-deoxyglucose was analyzed by ascending paper chromatography with two solvent systems. In these experiments, cell monolayers after incubation with 1 mM 2-deoxyglucose for 45 seconds were extracted with 0.5 N NaOH, neutralized with 1 N HCl and then were chromatographed (Tsan and Berlin, '71b). Only a small fraction of the radioactivity had the same Rf value as 2-deoxyglucose. The majority of the radioactivity was in one peak remaining a t the origin. Presumably, this peak represents 2-deoxyglucose-6-phosphate, since 2-deoxyglucose is phosphorylated by hexokinase (Romano and Colby, '73). There was no difference between PMN with and without phagocytosis; 6.7 +- 0.2% and 7.1 f 0.4% (mean f S.D., 3 experiments) remained as 2-deoxyglucose respectively. This small amount of 2-deoxyglucose is partly due to the extracellular contamination. Thus, practically all the transported 2-deoxyglucose was phosphorylated under these experimental situations. Effect ofphagocytosis on membrane transport in PMNfrompatients with chronic granulomatous disease Polymorphonuclear leukocytes from patients with chronic granulomatous disease
(CGD) are characterized by normal capacity for phagocytosis, but fail to show the stimulation of the hexose monophosphate shunt and the production of H,O, and superoxide normally associated with phagocytosis (Holmes e t al., '67; Babior, '78). Thus, CGD PMN ingest particles normally, but in contrast to normal PMN, there is no stimulation of glucose oxidation via the hexose monophosphate shunt during phagocytosis. The effect of phagocytosis on 2-deoxyglucose transport was studied in CGD PMN. As a reference, lysine transport was also determined. The results of these studies are shown in table 2. The rates of lysine and 2-deoxyglucose transport in CGD PMN remained unchanged after phagocytosis. DISCUSSION
In this study, I demonstrated t h a t the rates of 2-deoxyglucose transport in human PMN and rabbit AM were unchanged after phagocytosis of latex particles for 20 minutes. On the basis of the kinetic studies, and lack of exchange diffusion phenomenon in human PMN, i t is clear that 2-deoxyglucose transport system remains t h e same after phagocytosis. Since 2-deoxyglucose shares the same transport system as glucose, similar conclusion applies to the effect of phagocytosis on glucose transport system. This is in contrast to the marked stimulation of glucose oxidation associated with particle ingestion. Although it is possible that the number of glucose transport carriers may be reduced during the internalization of the plasma membrane which accompanies phagocytosis, and these reduced transport carriers are compensated by a n increased rate of transport due to exchange diffusion, two pieces of evidence suggest that this is unlikely. First, even after preloading with glucose, no exchange diffusion phenomenon was demonstrated. Second, the fate of intracellular 2-deoxyglucose was the same in
PHAGOCYTOSIS AND 2-DEOXYGLUCOSE TRANSPORT
human PMN with and without phagocytosis; i.e., almost all the transported 2-deoxyglucose was phosphorylated. This also explains why exchange diffusion phenomenon could not be demonstrated. Thus, the results suggest that the number of glucose transport carriers remains the same after phagocytosis. These observations confirm our previous findings (Tsan and Berlin, '71a) in five different transport systems. In our previous study we have also demonstrated that this constancy of the number of transport carriers after phagocytosis does not depend on the introduction of new transport carriers into the plasma membrane during phagocytosis (Tsan and Berlin, '71a). Therefore transport sites are preserved during phagocytosis, suggesting t h a t the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites. This concept of topographic separation of membrane functions has been repeatedly demonstrated. Griffin and Silverstein ('74) show that ingestion of one type of particle does not trigger generalized phagocytosis of all particles attached to the cell membrane. Noseworthy et al. ('72) also show that the amount of surface sialic acids are the same before and after phagocytosis. Since our original report of the effect of phagocytosis on membrane transport, two groups of investigators have repeated similar experiments. Dunham et al. ('74) reported that potassium and amino acid transports in human PMN after phagocytosis were reduced, and they attributed the difference between their results and those of ours to the different methods used. We used the rapid sampling technique of Hawkins and Berlin ('69) utilizing cell monolayers, while they used the cells in suspension. However, since they measured the uptake of substrates after ten minutes incubation which did not represent the initial rate of transport (this was quite obvious from their own results shown in figure 1 [Dunham et al., '7411, it is clear that they were not studying transport systems. In addition, PMN after ingesting many particles are easily traumatized by repeat pipetting, a process required in their washing procedure. In contrast, Bonventre et al. (Bonventre and Mukkada, '74; Straus et al., '77a,b) using monolayer technique, confirm our observation that amino acid transports remain the same after phagocytosis. One could argue that cells in suspension more closely resemble actual phys-
29
iological conditions than those adhering to surfaces. However, since the site of action of PMN is in the tissues rather than in circulation, i t seems quite likely that a n examination of cells adhering to surfaces could represent a close approximation to actual physiological conditions. Bonventre et al. (Bonventre and Mukkada, '74; Straus et al., '77a,b) also studied the glucose transport before and after phagocytosis, and reported that after phagocytosis, there was a 30-50%enhancement of 2-deoxyglucose transport in guinea pig and mouse peritoneal macrophages but not in PMN. In this study, I demonstrated that 2-deoxyglucose transport remained unaffected after phagocytosis in human PMN as well as rabbit alveolar macrophages. The reason for this discrepancy is not clear. It is tempting to suggest that the enhanced 2-deoxyglucose transport after phagocytosis in peritoneal macrophages as demonstrated by Bonventre et al. (Bonventre and Mukkada, '74; Straus et al., '77a,b) is due to exchange diffusion. However, I could not demonstrate any 'exchange diffusion phenomenon in human PMN. The experimental conditions in these two studies are also different. I used rabbit alveolar macrophages, whereas they utilized mouse and guinea pig peritoneal macrophages; I used latex particles to induce phagocytosis, whereas they used heat-killed staphylococci. In addition, they induced phagocytosis in the presence of glucose; the monolayers then were incubated in the balanced salt solution without glucose for 30 minutes before the rate of transport was determined. I induced phagocytosis in the absence of glucose. Stossel et al. ('70) have shown that in the absence of glucose, there is enhanced breakdown of cellular glycogen to support phagocytosis. Leukocytes from patients with chronic granulomatous disease ingest particles normally, but do not show the marked stimulation of glucose oxidation via the hexose monophosphate shunt normally associated with phagocytosis. However, they do have intact hexose monophosphate shunt enzyme systems (Holmes et al., '67). In this study, I demonstrated that CGD PMN also had normal 2-deoxyglucose transport before and after phagocytosis. ACKNOWLEDGMENTS
This work was supported by U.S. Public Health Service Research Grants AI-13004 and
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MIN-FU TSAN
GM-10548. Doctor Tsan is the recipient of a Research Career Development Award (AI00194) from National Institute of Allergy and Infectious Diseases. LITERATURE CITED Babior, B. M. 1978 Oxygen-dependent microbial killing of phagocytes. New Engl. J. Med., 298: 721-725. Bonventre, P. F.,and A. J. Mukkada 1974 Augmentation of glucose transport in macrophages after particle ingestion. Inf. Imm., 10: 1391-1396. Bayum, A 1968 Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest., 21 (Suppl. 97): 77-89. Dunham, P. B., I. M. Goldstein and G. Weissmann 1974 Potassium and amino acid transport in human leukocytes exposed to phagocytic stimuli. J. Cell Biol., 63: 215-226. Essner, E. 1960 An electron microscopic study of erythrophagocytosis. J. Biophys. Biochem. Cytol., 7: 329-334. Griffin, F. M., and S. C. Silverstein 1974 Segmental response of t h e macrophage plasma membrane to a phagocytic stimulus. J. Exp. Med., 139: 323-336. Hawkins, R. A,, and R. D. Berlin 1969 Purine transport in polymorphonuclear leukocytes. Biochim. Biophys. Acta, 173: 324-337. Heinz, E., and P. M. Walsh 1958 Exchanges diffusion, transport and intracellular level of amino acids in Ehrlich carcinoma cells. J. Biol. Chem., 233: 1488-1493. Holmes, B., A. R. Page and R. A. Good 1967 Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocyte function. J. Clin. Invest., 46: 1422-1432. Korn, E. D., and R . A. Weisman 1967 Phagocytosis of latex
beads by Acanthamoeba. 11.Electron microscopic study of initial events. J. Cell. Biol., 34: 219-227. Myrvik, Q. N., E. S. Leake and B. Fariss 1961 Studies on pulmonary alveolar macrophages from the normal rabbit. A technique t o procure them in a high state of purity. J. Immunol., 86: 128-132. Noseworthy, J., Jr., H. Korchak and M. L. Karnovsky 1972 Phagocytosis and the sialic acid of the surface of polymorphonuclear leukocytes. J. Cell. Physiol., 79: 91-96. Romano, A. H., and C. Colby 1973 SV-40 virus transformation of mouse 313 cells does not specifically enhance sugar transport. Science, 179: 1238-1241. Sbarra, A. J., and M. L. Karnovsky 1959 The biochemical basis of phagocytosis. I. Metabolic changes during the ineestion of Dartides bv DOlVnIOrDhOnUCk?ar leukocvtes. J . h o l . Che;., 234: 1355:13k2. Stossel, T. P., F. Murad, R. J. Mason and M. Vaughan 1970 Regulation of glycogen metabolism in polymorphonuclear leukocytes. J. Clin. Invest., 245: 6228-6234. Straus, D. C., J. G. Imhoff and P. F. Bonventre I977a Membrane transport of amino acid and hexose by guinea pig and mouse phagocytes. J. Reticuloendoth. SOC.,22: 403-416. 1977b Membrane transport by guinea pig peritoneal exudate leukocytes: Effect of phagocytosis on hexose and amino acid transport. J. Cell. Physiol., 93: 105-116. Tsan, M. F., and R. D. Berlin 1971a Effect of phagocytosis on membrane transport of non-electrolytes. J. Exp. Med., 134: 1016-1035. 1971b Membrane transport in the rabbit alveolar macrophage. The specificity and characteristics of amino acid transport systems. Biochim. Biophys. Acta., 241: 155-169. Tsan, M. F., B. Newman and P. A. McIntyre 1976 Surface sulfhydryl groups and phagocytosis-associated oxidative metabolic changes in human polymorphonuclear leukocytes. Brit. J. Haematol., 33: 189-204.
ABSTRACT 2-Deoxyglucose transport was characterized in human polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (AM). The Km was 1 mM for human PMN and 1.6 mM for rabbit AM, and the Vmax was pmoles/45 sec/106 AM. pmoles/45 sec/106 PMN and 5.09 x 0.66 x The rate of 2-deoxyglucose transport was the same before and after phagocytosis in PMN from normal individuals and three patients with chronic granulomatous disease, as well as rabbit AM. Studies of the kinetics of 2-deoxyglucose transport and intracellular fate of 2-deoxyglucose in human PMN indicate that the nature of the membrane transport system is not altered by phagocytosis. The results support the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites. Phagocytosis and carrier-mediated membrane transport are two distinct membrane functions by which substances are translocated across the plasma membrane. During phagocytosis, a portion of the plasma membrane is internalized (Essner, '60; Korn and Weisman, '67; Tsan and Berlin, '71a). We (Tsan and Berlin, '71a) have previously demonstrated that in five separate transport systems in rabbit polymorphonuclear leukocytes (PMN) (adenosine and two adenine transport systems) and alveolar macrophages (AM) (adenosine and lysine transport systems), the rate of transport was unaffected even after a large portion of the membrane had been internalized. We (Tsan and Berlin, '71a) provided evidence that transport sites (carriers) were preserved on the cell surface during internalization of the plasma membrane which accompanied phagocytosis. The results are consistent with the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites. Confirmatory observations have recently been reported by others (Straus et al., '77a,b). Phagocytosis is accompanied by a marked stimulation of glucose oxidation, especially through the hexose monophosphate shunt (Sbarra and Karnovsky, '59). This increased glucose utilization can be accomplished by J. CELL. PHYSIOL. (1979) 99: 23-30.
accelerated glycogen breakdown (glycogenolysis) or derived from extracellular source (Sbarra and Karnovsky, '59; Stossel e t al., '70). Therefore, I extend our previous observation to study the effect of phagocytosis on glucose transport utilizing 2-deoxy-D-glucose, a glucose analog. The results of this study form the basis of this report. MATERIALS AND METHODS
Chemicals I4C-D-glucose, 14C-2-deoxy-D-glucose and 14C-lysine (all uniformly labelled) were obtained from New England Nuclear, Boston, Massachusetts. Ficoll was obtained from Pharmacia Fine Chemicals, Inc., Uppsala, Sweden and sodium diatriazoate (Hypaque) from Winthrop Laboratories, New York. Polystyrene latex (0.79 pm in diameter) was obtained from the Dow Chemical Co., Indianapolis, Indiana. The preparation of leukocytes and alveolar macrophages Polymorphonuclear leukocytes were isolated from normal individuals and three patients with x-linked chronic granulomatous Received Oct. 12, '18.Accepted Nov. 16, '78. I Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; AM, alveolar macrophages; CGD, chronic panulomatous disease.
23
24
MIN-FU TSAN
disease (CGD) as described previously (Tsan et al., '76). Briefly, venous blood was obtained and leukocytes were isolated by dextran sedimentation of red blood cells, differential centrifugation and NH,Cl lysis of contaminating red cells. The leukocytes then were suspended in 10 ml of modified Hanks' solution (Tsan and Berlin, '71b) with 5 mM glucose, and placed on top of a 10-ml Ficoll-Hypaque mixture and centrifuged a t 400 g for 40 minutes a t 20°C according to Boyum ('68). The pellet which consisted of 97-99%pure PMN by differential counting was washed twice with modified Hanks' solution (5 mM glucose). Rabbit alveolar macrophages were obtained by the method of Myrvik et al. ('61) as modified by Tsan and Berlin ('71b). Cell monolayers used in all experiments were formed as previously described on round glass cover slips at a cell density of 2 million PMN and 0.3 million AM, per cover slip (Tsan and Berlin, '71a,b). Determination of membrane transport by a rapid-sampling technique The technique developed by Hawkins and Berlin ('69) was used to characterize the glucose transport system and study the effect of phagocytosis on glucose transport in human PMN and rabbit AM. After the formation of cell monolayers, the coverslips were pre-incubated with modified Hanks' solution without glucose for 20 minutes to deplete intracellular and remove extracellular glucose. The uptake of radioactive glucose in 45 seconds, which represents the initial rate of transport before and after phagocytosis, then was determined. This value was corrected for diffusion and extracellular contamination in order to obtain the carrier-mediated transport. The radioactivity recovered a f t e r incubation of monolayers a t 37°C a t supersaturating concentration of substrate (40 mM glucose) was considered to be the sum of diffusion and contamination components (Tsan and Berlin, '71b). In order to determine whether glucose and 2-deoxyglucose share the same transport system, competitive studies were carried out measuring the uptake of 0.1 mM ''CC-glucose or 0.1 mM 14C-2-deoxyglucosein the presence of 20 mM glucose or 2-deoxyglucose. These studies showed t h a t 2-deoxyglucose and glucose shared the same transport system, and subsequent studies were performed with 2-deoxyglucose only. The intracellular fate of 2-deoxyglucose was analyzed by ascending paper
chromatography with two solvent systems (tbutanol : methylethylketone : formic acid : H 2 0 , 40:30:15:15, and n-butanol : ethanol : HzO, 52:33: 15) as described previously (Tsan and Berlin, '71b). For the determination of membrane transport after phagocytosis, monolayers were first incubated with media containing 1%by weight of polystyrene latex particles (0.79 p in diameter) and 2 mM Mg++ in modified Hanks' solution without glucose for 20 minutes a t 37°C. Maximal phagocytosis was achieved under this experimental condition. At the end of the incubation, the coverslips were drained and washed with modified Hanks' solution at room temperature, then the rate of 2-deoxyglucose transport was determined. Controls were done with the media containing no particles and the rate of transport determined in the same way (Tsan and Berlin, '71a). All experiments were done with duplicate monolayers and the results averaged. The results are expressed as the amount of 2-deoxyglucose transported per 45 seconds per monolayer. RESULTS
The kinetics of 2-deoxyglucose transport in human PMNand rabbitAM Initial rates of 2-deoxyglucose were measured a t substrate concentrations between 0.1 mM and 4 mM. The radioactivity per coverslip was corrected for diffusion and extracellular contamination (Tsan and Berlin, '71b), and converted to wmoles of 2-deoxyglucose transported per monolayer. The data were expressed in a double reciprocal plot, from which the Km and Vmax were calculated. The derived values of Km and Vmax of 2-deoxyglucose in both cell types were quite constant. The results of typical experiments are shown in figure 1 for human PMN and figure 2 for rabbit AM. The Km was 1mM and the Vmax pmoles/45 sec/106cells was about 0.66 x for human PMN. The Km and Vmax for rabbit AM were 1.6 mM and 5.09 X lo-' pmoles/45 sec/106 cells respectively. These Km values are similar to those obtained in guinea pig peritoneal exudate PMN and macrophages (Straus et al., '77a,b; Bonventre and Mukkada, '74). Effect ofphagocytosis on 2-deoxyglucose transport The rate of 2-deoxyglucose transport was measured a t 1 mM substrate concentration
25
PHAGOCYTOSIS AND 2-DEOXYGLUCOSE TRANSPORT
l/C*"C.(tnM) Fig. 1 Kinetics of 2-deoxyglucose transport in human polymorphonuclear leukocytes. Cell monolayers were incubated in the presence of 'C-2-deoxyglucose in concentrations from 0.1 mM to 2 mM for 45 seconds. pmolesI45 sec/2 million cells. The initial rate (V)was expressed as
60
50
40
.>
\
-1
30
0
1
2 3 I/Conc. (mM)
4
5
Fig. 2 Kinetics of 2-deoxyglucose transport in rabbit alveolar macrophages. Cell monolayers were incubated in the presence of "C-2-deoxyglucose in concentrations from 0.2 mM to 4 mM for 45 seconds. The initial pmoles/45 sed0.3 million cells. rate (V)was expressed as
26
MIN-FU TSAN TABLE 1
Effect of phagocytosis on 2-deoxyglucose transport by human polymorphonuclear leukocytes and rabbit alveolar macrophages Rate of transport ' (?SE)
Polymorphonuclearleukocytes Alveolar macrophages
Control
Phagocytosis
7.0420.71(7)3 0.5320.04(7)
6.3520.82(7) 0.54-tO.07(7)
P values
> 0.40 >0.70
' The rate of transport was determined at 1 mM 2-deoxyglucose and expressed as (lo-' ,~molesJ45sec per 2 million cells) for p l y morphonuclear leukocytes and (10.' pmolesJ45 Bec per 0.3 million cells) for alveolar macrophages. P values were determined based on pair differences. Numbers in parentheses = number of experiments, each experiment was done with duplicate monolayers and results averaged.
after the cells were induced to phagocytize polystyrene latex particles for 20 minutes. The results are shown in table 1. It is obvious that the rate of 2-deoxyglucose transport was not significantly different after phagocytosis. The kinetics of carrier-mediated membrane transport follows the enzyme kinetics and the initial reaction is often represented by the following model (Tsan and Berlin, '71a), ki kz (a) C + S e s C S - C + S i k3
where C represents carrier, and Se and Si represent extracellular and intracellular substrate concentrations, respectively. CS represents the concentration of the carrier-substrate complex. The k's (kl, k 2 and k,) represent the rate constants. If Ct represents the total number of carriers, then the maximal transport rate can be expressed as: (b) Vmax
=
kz [CtSI.
Thus, Vmax is proportional to the total number of carriers under each experimental condition. The initial rate (V) of transport at a single concentration S is determined by the following equation:
From equation (c), i t is apparent that three factors can influence the initial rate, namely, k,, [CtSI, and Km. The number of carriers are reflected by the initial rates under various experimental conditions only when k 2 and km remain constant. Therefore, i t is of prime importance to find out whether these factors are changed by phagocytosis. It is assumed that in these experiments k, can be affected only by exchange diffusion (Tsan and Berlin, '71a).
Effect of phagocytosis on the kinetics of 2-deoxyglucose transport Kinetic studies of 2-deoxyglucose transport in human PMN with and without phagocytosis were carried out and the results as expressed by double reciprocal plot are shown in figure 3. The Km and Vmax remained the same after phagocytosis. Therefore the rates of transport shown in table 1, as determined a t a single substrate concentration, are satisfactory indices of the Vmax of the 2-deoxyglucose transport system. Effect of preloading with glucose on 2-deoxyglucose transport The stimulation of tracer influx by high intracellular substrate concentrations has been repeatedly described and designated exchange diffusion (Heinz and Walsh, '58; Tsan and Berlin, '71b). This is presumably because of an increase in k, in equation (c) (Tsan and Berlin, '71a). I t is possible that the intracellular concentration of glucose increased during phagocytosis due to enhanced glycogenolysis (Stossel et al., '70). Although the affinity of 2deoxyglucose for the carrier (Km) remains unchanged, the number of carrier may be reduced but compensated by an increased rate of transport because of exchange diffusion (i.e., k, increased) secondary to increased intracellular glucose concentration after phagocytosis. In order to rule out this possibility, human PMN were preloaded with 10 mM glucose for one to ten minutes before the rate of 2-deoxyglucose transport was determined. Preloading with 10 mM 2-deoxyglucose resulted in cell damage and PMN detached from the coverslips. If one assumes that the transported glucose is not metabolized, preloading with 10 mM glucose for ten minutes results in
PHAGOCYTOSIS AND 2-DEOXYGLUCOSE TRANSPORT
-
Control Phagocytosis
l/Conc.(rnM) Fig. 3 Kinetics of 2-deoxyglucose transport in human polymorphonuclear leukocytes with and without phagocytoais. Cell monolayers were first incubated with 1% polystyrene latex in the absence of glucose for 20 minutes, rinsed, and then 45-second uptake of “C-2-deoxyglucose in concentrations from 0.2 mM to 4 mM was measured. Control was incubated in the absence of particles. V was expressed as ~ m o l e s / 4 5secA million cells.
10
8
......w . * ............ )
c L
o
0
e..........................................
8””””’........
e
0
a 2 6
E
I
0 0)
4 0
c
a
a
Control
e Phagocytosis
2
Fig. 4 Effect of glucose-preloading on 2-deoxyglucoae transport by human polymorphonuclear leukocytes with and without phagocytosis. Cell monolayers were first incubated with 1%polystyrene latex, rinsed and then incubated with 10 m M glucoae for one to ten minutes. Cell monolayers were rinsed and the rate of 2-deoxyglucose transport was then determined. Control was incubated in the absence of particles. The rate of 2deoxyglucose transport was expressed a s pmoles/45 s e c h million cells.
27
28
MIN-FU TSAN TABLE 2
Effect ofphagocytosis on 2-deoxyglucose and lysine transport by polymorphonuclear leukocytes from three patients with chronicgranulomatous disease Rate of transport (&SE)
2-deoxyglucose Lysine
Concentration
Control
Phagocytosis
P value
1 mM 0.1mM
6.49k2.12(5)3 1.5650.34(3)
6.3951.41(5) 1.57-tO.25(3)
>0.80
' The rate of transport was expressed as (10.'
im1olesI45 sec per 2 million cells) for 2-deoxyglucose and
>0.60 pmolesl45 see per 2
million cells) for lysine. P values were determined based on pair differences. Numbers in parentheses = number of experiments. Each experiment was done in duplicate and results averaged.
'
an intracellular concentration of glucose of 2.5 mM (assuming PMN cell water of 0.346 pl/million cells) (Hawkins and Berlin, '69). However, this is not true since most of t h e transported glucose is metabolized (see below). As shown in figure 4, preloading with glucose did not affect the rate of P-deoxyglucose transport by human PMN with and without phagocytosis. Thus, there was no exchange diffusion phenomenon under these experimental conditions.
Intracellular fate of transported 2-deoxyglucose The fate of 2-deoxyglucose was analyzed by ascending paper chromatography with two solvent systems. In these experiments, cell monolayers after incubation with 1 mM 2-deoxyglucose for 45 seconds were extracted with 0.5 N NaOH, neutralized with 1 N HCl and then were chromatographed (Tsan and Berlin, '71b). Only a small fraction of the radioactivity had the same Rf value as 2-deoxyglucose. The majority of the radioactivity was in one peak remaining a t the origin. Presumably, this peak represents 2-deoxyglucose-6-phosphate, since 2-deoxyglucose is phosphorylated by hexokinase (Romano and Colby, '73). There was no difference between PMN with and without phagocytosis; 6.7 +- 0.2% and 7.1 f 0.4% (mean f S.D., 3 experiments) remained as 2-deoxyglucose respectively. This small amount of 2-deoxyglucose is partly due to the extracellular contamination. Thus, practically all the transported 2-deoxyglucose was phosphorylated under these experimental situations. Effect ofphagocytosis on membrane transport in PMNfrompatients with chronic granulomatous disease Polymorphonuclear leukocytes from patients with chronic granulomatous disease
(CGD) are characterized by normal capacity for phagocytosis, but fail to show the stimulation of the hexose monophosphate shunt and the production of H,O, and superoxide normally associated with phagocytosis (Holmes e t al., '67; Babior, '78). Thus, CGD PMN ingest particles normally, but in contrast to normal PMN, there is no stimulation of glucose oxidation via the hexose monophosphate shunt during phagocytosis. The effect of phagocytosis on 2-deoxyglucose transport was studied in CGD PMN. As a reference, lysine transport was also determined. The results of these studies are shown in table 2. The rates of lysine and 2-deoxyglucose transport in CGD PMN remained unchanged after phagocytosis. DISCUSSION
In this study, I demonstrated t h a t the rates of 2-deoxyglucose transport in human PMN and rabbit AM were unchanged after phagocytosis of latex particles for 20 minutes. On the basis of the kinetic studies, and lack of exchange diffusion phenomenon in human PMN, i t is clear that 2-deoxyglucose transport system remains t h e same after phagocytosis. Since 2-deoxyglucose shares the same transport system as glucose, similar conclusion applies to the effect of phagocytosis on glucose transport system. This is in contrast to the marked stimulation of glucose oxidation associated with particle ingestion. Although it is possible that the number of glucose transport carriers may be reduced during the internalization of the plasma membrane which accompanies phagocytosis, and these reduced transport carriers are compensated by a n increased rate of transport due to exchange diffusion, two pieces of evidence suggest that this is unlikely. First, even after preloading with glucose, no exchange diffusion phenomenon was demonstrated. Second, the fate of intracellular 2-deoxyglucose was the same in
PHAGOCYTOSIS AND 2-DEOXYGLUCOSE TRANSPORT
human PMN with and without phagocytosis; i.e., almost all the transported 2-deoxyglucose was phosphorylated. This also explains why exchange diffusion phenomenon could not be demonstrated. Thus, the results suggest that the number of glucose transport carriers remains the same after phagocytosis. These observations confirm our previous findings (Tsan and Berlin, '71a) in five different transport systems. In our previous study we have also demonstrated that this constancy of the number of transport carriers after phagocytosis does not depend on the introduction of new transport carriers into the plasma membrane during phagocytosis (Tsan and Berlin, '71a). Therefore transport sites are preserved during phagocytosis, suggesting t h a t the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites. This concept of topographic separation of membrane functions has been repeatedly demonstrated. Griffin and Silverstein ('74) show that ingestion of one type of particle does not trigger generalized phagocytosis of all particles attached to the cell membrane. Noseworthy et al. ('72) also show that the amount of surface sialic acids are the same before and after phagocytosis. Since our original report of the effect of phagocytosis on membrane transport, two groups of investigators have repeated similar experiments. Dunham et al. ('74) reported that potassium and amino acid transports in human PMN after phagocytosis were reduced, and they attributed the difference between their results and those of ours to the different methods used. We used the rapid sampling technique of Hawkins and Berlin ('69) utilizing cell monolayers, while they used the cells in suspension. However, since they measured the uptake of substrates after ten minutes incubation which did not represent the initial rate of transport (this was quite obvious from their own results shown in figure 1 [Dunham et al., '7411, it is clear that they were not studying transport systems. In addition, PMN after ingesting many particles are easily traumatized by repeat pipetting, a process required in their washing procedure. In contrast, Bonventre et al. (Bonventre and Mukkada, '74; Straus et al., '77a,b) using monolayer technique, confirm our observation that amino acid transports remain the same after phagocytosis. One could argue that cells in suspension more closely resemble actual phys-
29
iological conditions than those adhering to surfaces. However, since the site of action of PMN is in the tissues rather than in circulation, i t seems quite likely that a n examination of cells adhering to surfaces could represent a close approximation to actual physiological conditions. Bonventre et al. (Bonventre and Mukkada, '74; Straus et al., '77a,b) also studied the glucose transport before and after phagocytosis, and reported that after phagocytosis, there was a 30-50%enhancement of 2-deoxyglucose transport in guinea pig and mouse peritoneal macrophages but not in PMN. In this study, I demonstrated that 2-deoxyglucose transport remained unaffected after phagocytosis in human PMN as well as rabbit alveolar macrophages. The reason for this discrepancy is not clear. It is tempting to suggest that the enhanced 2-deoxyglucose transport after phagocytosis in peritoneal macrophages as demonstrated by Bonventre et al. (Bonventre and Mukkada, '74; Straus et al., '77a,b) is due to exchange diffusion. However, I could not demonstrate any 'exchange diffusion phenomenon in human PMN. The experimental conditions in these two studies are also different. I used rabbit alveolar macrophages, whereas they utilized mouse and guinea pig peritoneal macrophages; I used latex particles to induce phagocytosis, whereas they used heat-killed staphylococci. In addition, they induced phagocytosis in the presence of glucose; the monolayers then were incubated in the balanced salt solution without glucose for 30 minutes before the rate of transport was determined. I induced phagocytosis in the absence of glucose. Stossel et al. ('70) have shown that in the absence of glucose, there is enhanced breakdown of cellular glycogen to support phagocytosis. Leukocytes from patients with chronic granulomatous disease ingest particles normally, but do not show the marked stimulation of glucose oxidation via the hexose monophosphate shunt normally associated with phagocytosis. However, they do have intact hexose monophosphate shunt enzyme systems (Holmes et al., '67). In this study, I demonstrated that CGD PMN also had normal 2-deoxyglucose transport before and after phagocytosis. ACKNOWLEDGMENTS
This work was supported by U.S. Public Health Service Research Grants AI-13004 and
30
MIN-FU TSAN
GM-10548. Doctor Tsan is the recipient of a Research Career Development Award (AI00194) from National Institute of Allergy and Infectious Diseases. LITERATURE CITED Babior, B. M. 1978 Oxygen-dependent microbial killing of phagocytes. New Engl. J. Med., 298: 721-725. Bonventre, P. F.,and A. J. Mukkada 1974 Augmentation of glucose transport in macrophages after particle ingestion. Inf. Imm., 10: 1391-1396. Bayum, A 1968 Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest., 21 (Suppl. 97): 77-89. Dunham, P. B., I. M. Goldstein and G. Weissmann 1974 Potassium and amino acid transport in human leukocytes exposed to phagocytic stimuli. J. Cell Biol., 63: 215-226. Essner, E. 1960 An electron microscopic study of erythrophagocytosis. J. Biophys. Biochem. Cytol., 7: 329-334. Griffin, F. M., and S. C. Silverstein 1974 Segmental response of t h e macrophage plasma membrane to a phagocytic stimulus. J. Exp. Med., 139: 323-336. Hawkins, R. A,, and R. D. Berlin 1969 Purine transport in polymorphonuclear leukocytes. Biochim. Biophys. Acta, 173: 324-337. Heinz, E., and P. M. Walsh 1958 Exchanges diffusion, transport and intracellular level of amino acids in Ehrlich carcinoma cells. J. Biol. Chem., 233: 1488-1493. Holmes, B., A. R. Page and R. A. Good 1967 Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocyte function. J. Clin. Invest., 46: 1422-1432. Korn, E. D., and R . A. Weisman 1967 Phagocytosis of latex
beads by Acanthamoeba. 11.Electron microscopic study of initial events. J. Cell. Biol., 34: 219-227. Myrvik, Q. N., E. S. Leake and B. Fariss 1961 Studies on pulmonary alveolar macrophages from the normal rabbit. A technique t o procure them in a high state of purity. J. Immunol., 86: 128-132. Noseworthy, J., Jr., H. Korchak and M. L. Karnovsky 1972 Phagocytosis and the sialic acid of the surface of polymorphonuclear leukocytes. J. Cell. Physiol., 79: 91-96. Romano, A. H., and C. Colby 1973 SV-40 virus transformation of mouse 313 cells does not specifically enhance sugar transport. Science, 179: 1238-1241. Sbarra, A. J., and M. L. Karnovsky 1959 The biochemical basis of phagocytosis. I. Metabolic changes during the ineestion of Dartides bv DOlVnIOrDhOnUCk?ar leukocvtes. J . h o l . Che;., 234: 1355:13k2. Stossel, T. P., F. Murad, R. J. Mason and M. Vaughan 1970 Regulation of glycogen metabolism in polymorphonuclear leukocytes. J. Clin. Invest., 245: 6228-6234. Straus, D. C., J. G. Imhoff and P. F. Bonventre I977a Membrane transport of amino acid and hexose by guinea pig and mouse phagocytes. J. Reticuloendoth. SOC.,22: 403-416. 1977b Membrane transport by guinea pig peritoneal exudate leukocytes: Effect of phagocytosis on hexose and amino acid transport. J. Cell. Physiol., 93: 105-116. Tsan, M. F., and R. D. Berlin 1971a Effect of phagocytosis on membrane transport of non-electrolytes. J. Exp. Med., 134: 1016-1035. 1971b Membrane transport in the rabbit alveolar macrophage. The specificity and characteristics of amino acid transport systems. Biochim. Biophys. Acta., 241: 155-169. Tsan, M. F., B. Newman and P. A. McIntyre 1976 Surface sulfhydryl groups and phagocytosis-associated oxidative metabolic changes in human polymorphonuclear leukocytes. Brit. J. Haematol., 33: 189-204.
