plasma membrane synthesis in the macrophage following phagocytosis … · tion in cell volume,...

THE JOURNAL OB BIOLOGICAL CHE~STRY Vol. 247, No. 8, Issue of April 25, pp. 2439-2446, 1972

Printed in U.S.A.

Plasma Membrane Synthesis in the Macrophage following

Phagocytosis of Polystyrene Latex Particles*

ZENA WE~B$ AND ZANVIL A. COHN

From The Rockefeller University, New York, New York 10061

(Received for publication, October 22, 1971)

SUMMARY

Phagocytosis of polystyrene latex beads by mouse peritoneal macrophages results in the interiorization of large amounts of plasma membrane to form the phagolysosomal membrane. The isolated membrane of the phagolysosomes is initially similar to the plasma membrane in cholesterol and phospholipid content, and carries 5’-nucleotidase activity with the characteristics of the plasma membrane marker enzyme. Immediately after ingestion, the amount of the plasma membrane has descreased as judged by a reduction in cell volume, spreading, pinocytosis, and phagocytosis. During the next 6 hours, the phagolysosomal5’-nucleotidase activity decreases with a half-life of about 2 hours. Total cellular 5’-nucleotidase also decreases to a degree accounted for by the decrease in the phagolysosomal enzyme and in proportion to the amount of latex interiorized. After a lag of about 6 hours, cellular levels of the membrane constituents cholesterol and phospholipid begin to increase. The final net increase is linearly proportional to the amount of latex initially ingested and to the amount of plasma membrane interiorized. Only at this time do the macrophages begin to spread out on the glass surface, resume pinocytosis, and regain the ability to phagocytize a new test particle. The cellular levels of 5’-nucleotidase also begin to increase reaching control levels by 10 to 12 hours after phagocytosis. This activity is not associated with the phagolysosomes. These changes suggest the net synthesis of plasma membrane to replace the membrane interiorized during phagocytosis. This synthesis requires the presence of exogenous cholesterol molecules for use in the membrane. In the absence of cholesterol, plasma membrane functions are not restored and 5’-nucleotidase activity does not return to control levels. The inhibition of protein and RNA synthesis after phagocytosis blocks the net increment in cellular cholesterol, phospholipid, and 5’-nucleotidase activity.

Previous studies from this laboratory on cholesterol metabo- lism in the macrophage have indicated that free cholesterol is

* This investigation was supported in part by Research Grants AI 07012 and AI 01831 from the United States Public Health Serv- ice.

1 Current address, Strangeways Research Laboratory, Wort’s Causeway, Cambridge, England. Address reprint requests to The Rockefeller University.

found almost exclusively in plasma membrane and lysosomal membrane (1). Macrophages cultivated in vitro contain about one-third their cholesterol in lysosomal membrane and the remaining two-thirds in plasma membrane. Since cholesterol is not biosynthesized by the macrophage, it is likely that membrane turnover depends on the cholesterol exchanged from serum lipoproteins. Increases in total free cholesterol and phospholipid levels occur after extensive pinocytosis and phagocytoses of nondigestible substances (2). The increases in these membrane components result from the storage of many secondary lysosomes which are derived from plasma membrane during endocytosis. These changes suggested that the synthesis of cellular membrane had occurred. Studies from other laboratories have reported increased turnover of phospholipids during phagocytosis but have not shown net increases in the phospholipid content of phago- cytizing cells (3-6).

This communication reports our studies on the net synthesis of membrane following phagocytosis of polystyrene latex particles, and describes the transient presence of 5’-nucleotidase (5’-ribonucleotide phosphohydrolase EC 3.1.3.5), a plasma membrane marker enzyme (7) on the lysosomal membrane after extensive phagocytosis.

EXPERIMENTAL PROCEDURE

Harvesting and Cultivation of Cells

Homogeneous populations of mouse peritoneal macrophages were obtained from Rockefeller strain NCS mice, then cultivated in 15-cm2 glass T-flasks for biochemical studies; loo-mm plastic Petri dishes (Vangard International Inc., Red Bank, K. J.) for fractionation studies; or on glass coverslips for morphological studies, in Medium 199 (Microbiological Associates, Bethesda, Md.) supplemented with 20% newborn calf serum (NBC& Grand Island Biological Co., Grand Island, N. Y.) as described previously (1, 8). The complete medium is referred to as 207, NBCS. For biochemical measurements, macrophages were harvested from flasks in distilled water.

For phagocytosis experiments, polystyrene latex particles of diameter 1.099 pm (Dow Chemical Co., Midland, Mich.) were washed in Medium 199, resuspended in 20% NBCS at 500 pg per ml, and added to macrophages which had been cultivated for 24 hours. The cultures were exposed to latex for 60 min at 37”, washed twice with Medium 199, and then incubated in fresh 20% NBCS.

Cell Fractionation

Isolation of Mouse Macrophage Latex Phagolysosomes- Phago- lysosomes were prepared by a modified procedure of Wetzel

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2440 Membrane Synthesis in Macrophages after Phagocytosis Vol. 247, No. 8

and Korn (9). Macrophages were grown in 20y0 NBCS for 24 hours. Latex particles of diameter 1.099 pm were resuspended in 20% NBCS at 500 fig per ml (about 200 to 1000 beads per cell), and added to the cultures. The cells and beads were incubated at 37” for 60 min and the cells were rinsed twice with Medium 199 to remove extracellular beads. Cells were harvested immediately, or incubated for an additional period of time in fresh serum medium. Cells were harvested by scraping into phosphate-buffered 0.9% NaCl solution, concentrated by centrifugation at 500 x g for 5 min, resuspended in 3 ml of 30% sucrose (w/v), and homogenized in a 7-ml Dounce homogenizer. When about 90% cell breakage was achieved (phase contrast microscopy), 2.5 ml were placed in a tube, overlaid with 6 ml of 20% and 2.5 ml of 10% sucrose, and centrifuged at 100,000 X g for 60 min in the SW 41 rotor of the Beckman L-2 ultracentrifuge. Phagolysosomes were concentrated at the 10 to 20% sucrose interface (IF). 5’-Nucleotidase was assayed in fractions diluted with water after a single cycle of freezing and thawing. Lyso- somal enzymes were assayed after freezing and thawing six times to dest’roy latency.

Preparation of Phagolysosomal Membrane-The procedure used was based on the principle of destroying the latency of the phagolysosomes so that soluble contents leak out, followed by reisolation of the latex particles with their adherent membranes. With [7JH]cholesterol as a membrane marker (1, 2), this method resulted in no loss of membrane while the contents were depleted of protein and acid hydrolases. Phagolysosomes from the lo/20 IF were frozen and thawed three times, diluted with 5 volumes of water, and centrifuged at 1000 X g for 20 min in a Lourdes centrifuge. The pellet contained latex plus membranes. Fur- ther dilution and centrifugation steps gave little additional purification.

Isolation of Macrophage Plasma Membrane-Membranes were isolated by the Tris method of Warren et al. (10) modified for use with macrophages. Macrophages grown in vitro for 24 hours were placed at 4” for 60 min to round the cells, rinsed once with Medium 199, and then with 0.05 M Tris, pH 7.4, concentrated by centrifugation, and allowed to swell for 10 min at 4” in 2.7 ml of Tris plus 0.3 ml of 0.05 M MgC12. They were then broken with a Vortex mixer. The “homogenate” was mixed with 3.0 ml of 60% sucrose (0.005 M in MgCl*), 5.5 ml layered over 5 ml of 45y0 sucrose, and centrifuged at 2000 x g for 20 min. The membrane-enriched fraction recovered at the interface was diluted in 20% sucrose, and concentrated at 6000 X g for 20 min. No further purification was obtained by centri- fuging the membrane fraction to isopycnic equilibrium on a continuous sucrose density gradient. Morphological analysis of the fraction revealed the predominance of long strips of plasma membrane and occasional secondary lysosomes. The fraction was devoid of nuclei and contained only rare profiles of mito- chondria and rough endoplasmic reticulum.

Enzyme Assays

5’-Nucleotidase-This enzyme was assayed by the liberation of inorganic phosphate with 5 InM 5’-AMP as substrate at pH 8.5 in 10 mM Tris buffer in the presence of 10 mM Mg2+ according to the method described by Widnell and Unkeless (11). The pH curves were determined in 0.1 M barbital-acetate buffer. Ac- tivity was also assayed with 0.10 mM 5’-AMP at pH 7.9 with adenosine deaminase present in the reaction mixture as described by Belfield and Goldberg (12).

Acid Phosphatase-Acid phosphatase was assayed with 0.01 M

cr-naphthol acid phosphate (Dajac Laboratories, Philadelphia, Pa.) at pH 5.0 in 0.1 M acetate buffer as described by Axline (13).

Analytic Procedures

Protein-Protein was determined by the method of Lowry et al. with egg white lysosyme as standard.

Phospholipid Phosphorus- Lipids were extracted into chloro- form-methanol with the procedure of Folch et al. (14). The lipid phase was taken to dryness and the phospholipids were ashed with Mg(NO& by the method of Chen et al. (15). Inor- ganic phosphorus was determined with both inorganic phosphate and ashed dipalmitoyl lecithin (Mann Research Lab., New York) as standards, as described by Ames and Dubin (16).

Cholesterol-Cholesterol was assayed by gas-liquid chroma- tography (1) with 5-ol-cholestane as the internal standard.

Polystyrene Latex-This was assayed by a modification of the procedure described by Weisman and Korn (17). One volume (0.1 ml) of the aqueous sample containing the latex particles was mixed with 19 volumes of dioxane analytical reagent grade, and allowed to stand for 5 min. Absorption at 259 nm was determined, and compared to standards diluted from the pur- chased stock of 100 mg per ml. To prevent interference samples containing sucrose were diluted with water to final concentrations of sucrose less than 10% (w/v).

Materials

Polystyrene latex particles, 1.96 pm in diameter, were obtained from Dow Chemical Co., Midland, Mich.; bovine serum albumin, Fraction V, Armour Pharmaceutical Co., Chicago, Ill.; puromycin dihydrochloride, Nutritional Biochemical Corp., Cleve- land, 0.; bromotubercidin was the kind gift of Dr. E. Reich, The Rockefeller University. All other chemicals were commer- cial preparations of analytic reagent grade.

RESULTS

Cell Fractionation Xtudies

Preliminary studies were performed to study the composition of macrophage plasma membrane and phagolysosomal membrane, and to determine the amount of plasma membrane interiorized by phagocytosis.

Analysis of Latex Phagolysosomes

Macrophages which have been exposed to l.l-pm latex particles for 1 hour contain at least 100 particles per cell. Intracellu- larly, the latex particles are surrounded by unit membranes and contents which are acid phosphatase-positive (18). Results of an experiment in which macrophages were fractionated immediately after phagocytosis is shown in Table I. Cholesterol and phospholipid were enriched in the lo/20 IF fraction which contained most of the latex phagolysosomes as judged by morphol- ogy and latex recovery. With the values of this fraction to cor- rect for latex found in other fractions, phagolysosomes accounted for 68% of the cell cholesterol, 84% of the acid phosphatase, and 59% of the S’nucleotidase. In Table II the data for the lo/20 IF phagolysosomes are compared with those from other cell fractions. Cholesterol, acid phosphatase, and 5’nucleotidase were enriched in the phagolysosomes isolated immediately after phagocytosis. The amount of 5’-nucleotidase recovered in this


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TABLE I Isolation of latex phagolysosomes from macrophages

Phagolysosomes were isolated after a l-hour exposure to 500 rg mental Procedure.” Homogenate values (lOO’%) were: 52.3 pg of of polystyrene latex particles per ml of 2070 NBCS as described cholesterol; 172 nmoles of phospholipid; 2.72 mg of protein; 570 under “Experimental Procedure.” Fractions consisting of the units of acid phosphatase; 251 units of 5’-nucleotidase (a); 149 10% sucrose layer, the lo/20 IF layer which contained most of the units of 5’-nucleotidase (b). Enzyme units are nanomoles of sub- lat.ex, the 20% sucrose layer, the 20/30 IF fraction which also con- strate hydrolyzed per min; specific activity (S.A.) units are nano- tained a small band of latex, the 30yo sucrose layer which con- moles per min per mg of protein. 5’-Nucleotidase (a) was assayed tained pieces of membrane when monitored with phase contrast by the method of Widnell and Unkeless (11); 5’-nucleotidase (b) illumination, and the pellet resuspended in 10% sucrose were re- was assayed by the method of Belfield and Goldberg (12). n.d., moved from the gradient and assayed as described under “Experi- not deteimined.

I- Acid phosphatase S’-Nucleotidase (a) 5’.Nucleotidase (b) Cholesterol Phospholipid Protein

Fraction .I-

Per cent Per cent Per cent Per cent Per cent S.A. Per cent S.A Per cent S.A.

Homogenate. ... 10% ............ lo/20 IF ........ 20%. ........... 20/30 IF ........ 30%. ........... Pellet. .........

100 100 100 100 100

0 n.d. 0 3.7 0

37.2 33 15.0 50.6 42.3 0.3 n.d. 4.9 4.2 3.8

11.2 14 9.6 12.8 12.1 20.1 23 17.0 10.3 11.6 37.6 41 47.4 24.2 31.1

210

593 163 721 136 138

100 0

29.8 0.6 9.2

36.4 27.2

100 n.d. 34.7

n.d. 8.0

20.9 23.5

Recoveries (To). 106 110 94 106 101 103 87

55

128

43 68 27

- -

TABLE II Localization of 6’-nucleotidase and cholesterol in plasma membrane and lysosomal fractions

Cell fractions were obtained as described under “Experimental Procedure.”

Cholesterol I 5’.Nucleotidase I Acid phosphatase Latex

Fraction

Recoverya Cholesterol Cholesterol per protein per phospholipid Recoverya

Relative specific

activity Recovery~

Relative specific

activity Recovery

% Irg/w

Whole cells. 100 12 f 18 Phagolysosomes 25-48 (9)b 44 f 19 Phagolysosomal membrane. 27-360(3) 81 =t 27 Plasma membrane. . . / 13%25(6) j 51 f 21

moles/mole % %

0.69 f 0.12(7) 100 (8 ) 1 100 (7 )b 0.86 f 0.21(3) 21-36(5) 2.7(1.6p4.1)c 35-84(6)

Not done 25&31n(2) 3.6 (2.8-4.4) 8%180(3) 1.09 f 0.31(5) 32%48(2) 4.3(3.1-5.5) 8-12 (2)

1 3.3(2.1-5.5)” 1.8(1.2-2.6) 0.8(0.7-0.9)

%

100 (6 ) 33-91(9) 48~86~ (3)

- a Percentage of homogenate value recovered in this fraction. b Number of experiments in parentheses. c Range of values in parentheses.

fraction was proportionate to the amount of latex ingested, and could be used to estimate the percentage of plasma membrane interiorized. Phagolysosomes isolated 2 to 24 hours after phagocytosis contained about the same levels of cholesterol and acid phosphatase, but decreasing levels of 5’-nucleotidase. This point is discussed later. Almost all the acid phosphatase could be accounted for in latex-containing fractions. Acid cholesterol esterase (19) was also found in the phagolysosomes. This indicated that the discharge of previously existing lysosomes into the phagocytic vacuoles was nearly complete.

Analysis of Phagolysosomal Membrane

When phagolysosomal membrane was prepared by exposing the latex phagolysosomes to hypotonic shock, most of the acid phosphatase activity was lost, while cholesterol and 5’-nucleotidase activity were unchanged (Table II). The loss of protein from the phagolysosomes resulted in increased specific activity of 5’-nucleotidase.

Analysis of Plasma Membrane Fractions

Plasma membrane preparations isolated from macrophages which had not ingested latex contained large membrane sheets as well as smaller vesicles when examined microscopically, and accounted for about 10% of the cell protein (Table II). Up to 25% of the cholesterol was recovered, and the cholesterol to phospholipid molar ratio approached 1.0. 5’-Nucleotidase was enriched almost B-fold in some preparations. From kinetic experiments (2), it can be calculated that plasma membrane accounts for two-thirds of the cellular cholesterol. Thus, about 25 to 40% of the plasma membrane was recovered in these fractions. Con- tamination with lysosomes was minor in view of the low levels of acid phosphatase. Hence, in nonphagocytizing cells, essentially all the 5’-nucleotidase activity could be accounted for by the plasma membrane. Similar observations have also been made for alveolar macrophage plasma membrane and phago- 1ysosomes.l

1 Z. Werb, unpublished observations.


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TABLE III

Substrate specijicity of 5’-nucleotidase

Enzyme activity was assayed at pH 8.5 in 0.05 M Tris buffer which contained 10 mM MS++. Substrates were present in the reaction mixture at a final concentration of 5.0 mM. Addition of 20 mM EDTA to the reaction mixture inhibited substrate hydrolysis >95yo in all cases.

I Percentage of hydrolysis of Y-AMP Substrate

Homogenate

5’-AMP. 5’-UMP 5’-dAMP 2’,3’-AMP, 5’.TMP fi-Glycerophosphate

%

100 122 33

<1 90

< 1

1 ?hagolysosomes

TABLE IV

lp %

100 105 30

<1 84

<l

lasma membrane

%

100 87 25

<1 100 <1

E$ect of inhibitors on 5’-nucleotidase activity

5’-Nucleotidase activity was assayed at pH 5.0 and 8.5 in 0.05 M barbital-acetate buffer with 5 mM 5’-AMP as substrate. The reaction mixture contained 10 mM Mg++. Inhibitors were added to the final concentrations given below. Zn* (0.1 mM) and EDTA (0.5 mM) were mixed prior to addition to the reaction mixture. Enzyme activity is expressed as percentage of the appropriate control without inhibitor.

Inhibitor

None .................... Tartrate, 10 mM ......... Fluoride, 10 mM. ....... EDTA, 30 mM. ......... Zn++, 0.1 mM ........... Zn++, 0.1 mM, and EDTA,

0.5 rnM. .............. -

Enzyme activity, percentage of control

Homogenate Phagolysosomes

pH 5.0 pH 8.5

% % % % 100 100 100 100

7 103 12 95 18 45 12 51 20 8 35 21

105 0 108 8

58 109 65 103

pH 5.0 pH 8.5

Properties of Macrophage 5’-Nucleotidase

Stability--Experiments were performed to characterize the 5’nucleotidase activity found in the mouse macrophage. This enzyme was labile to freezing and thawing and 60% of the activity was lost after six cycles of freezing and thawing. Most assays were performed immediately after a single cycle of freezing and thawing. Specific activities of 30 to 120 nmoles per min per mg of protein were found in whole macrophage lysates from

control cultures when assayed by the method of Widnell and Unkeless (11). The values were consistent for a given set of cultures.

Substrate Speci$city--Macrophage homogenate, latex phagolysosomes isolated immediately after phagocytosis, and macrophage plasma membranes were studied to determine the substrate specificity of the 5’-nucleotidase activity (Table III). At pH 8.5 only 5’-nucleotides served as substrates. The enzyme specificity was identical in all fractions tested. No non- specific phosphatase activity was found. Enzyme activity was

4-

3-

48

4

*t 0

, I I I

IO 20 30 40 pg latex phagocytized in one hour

0

FIG. 1. Dose response of the increase in cholesterol content of macrophages 24 hours after phagocytosis of l.l-pm latex particles. Macrophage flask cultures wele exposed to latex particles at 0, 250, 500, and 100 pg per ml in 20% NBCS for 1 hour at 37”, then incubated for 24 hours in ZOY, NBCS. Ten micrograms of latex = 1 X 10’ particles.

inhibited >90% by 20 mM EDTA present in the reaction mixture.

Inhibitor Studies-The activity of whole cell lysates and phagolysosomes showed a peak at pH 5.0 and another between pH 7.2 and 9.5. The identity of the peaks was studied by using tartrate as an inhibitor of acid phosphatase (20), Znzf as an inhibitor of 5’-nucleotidase (21, 22), and fluoride ions as a general phosphatase inhibitor (23, 24). The data for pH 5.0 and 8.5 are shown in Table IV. The acid phosphatase activity was clearly differentiated from 5’-nucleotidase activity in both homogenate and phagolysosomes. In addition Znzf inhibition of 5’nucleotidase activity, could be completely reversed by EDTA. It was of interest that acid nucleotidase activity, which unlike 5’-nucleotidase, is not known to have a divalent cation requirement, was inhibited 50% by EDTA.

Increase in Cholesterol and Phospholipid after Phagocytosis

Dose Response-Initial experiments (2) had indicated that a net increase in cellular free cholesterol occurred in macrophages following phagocytosis of latex. This increase was studied as a function of particle number ingested at 24 hours after phagocytosis (Fig. 1). The increase was found to be a linear function of the amount of latex ingested.

Time Course of Cholesterol and Phospholipid Increases-Fol- lowing phagocytosis of latex particles, macrophage-free cholesterol and phospholipid increased in parallel, to about 160% of control cells which had not engaged in phagocytosis. This increase took place after a lag period of 4 to 8 (6.4 f 1.4 hours) for eight experiments. In Fig. 2a is shown the time course for one experiment. These increases were not due to increased number or size of the cells (25), but to an increased concentration of cholesterol-rich membrane in the macrophage (2). The increase in the cholesterol to protein ratio (Fig. 26) is a sensitive measure of cellular membrane concentration in these cells. In this experiment a 50% increase in the ratio occurred after a lag period of 6 hours.

Resumption of Phagocytosis and Pinocytosis after Extensive Phagocytosis-In amoebae and macrophages the rate of pinocytosis is decreased after rapid and extensive pinocytosis or


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(a)

”

Y 2 -------_ _ _ Contro

i

i

Latex

0 0 5 IO 15 20 25

(b)

Latex

0 (r i P Contro

IO I I

0 5 IO 15 20 25

Hours after phagocytosis

FIG. 2. Time course of increase of cholesterol and phospholipid content of macrophagei following phagocytosis of 1.1~pm latex particles. a, increase in cellular cholesterol and phospholipid in control and latex-laden cells during incubation in 20% NBCS. The latex- laden cells contained 41 i 6 pg of latex per flask. b, increase in the cholesterol to protein ratio after latex phagocytosis.

phagocytosis (26, 27). Resumption of pinocytosis and phago- pytosis, and spreading of macrophages were studied as measures of plasma membrane function following the phagocytosis of latex. Immediately after phagocytosis, macrophages were rounded and the amount of peripheral cytoplasm was small. About 5 hours after phagocytosis, the cells began to respread, and by 5 to 12 hours had well developed cytoplasmic veils with the phagolysosomes arranged in the “hof” of the nucleus. The rate of pinocytic vacuole formation and the ability to phagocytize a new test particle (1.96.pm latex particles) recovered in parallel, and approached control levels by 8 hours (Fig. 3). The recovery seen early after phagocytosis reflected heterogeneity of phagocytosis by macrophages. Cells which had ingested few 1.1~pm particles spread more readily and were able to phagocytize and pinocytize at early times. The recovery of these plasma membrane functions closely paralleled the increases in cholesterol and phospholipid content (Figs. 1, 2).

Effect of Phagocytosis of Latex Particles on Cellular Levels of 5’-Nucleotidase

Following the ingestion of latex particles, it was noticed that total cellular 5’-nucleotidase decreased up to 5 hours after phagocytosis. Moreover, the amount of 5’-nucleotidase recovered in the phagolysosomes also decreased. Cellular 5’-nucleotidase levels after phagocytosis were examined in detail for a range of values of latex ingested and compared with changes in cholesterol and phospholipid (Fig. 4). 5’-Nucleotidase decreased after phagocytosis reaching a minimum value at about 6 hours after ingestion. The magnitude of the decrease in activity was a function of the amount latex taken up by each culture. Then between 6 and 10 hours both the total cellular activity and its specific activity began to increase approaching control levels.

By 24 hours, control levels of 5’-nucleotidase had been achieved in all the cultures. The time of recovery paralleled the increases in cholesterol and phospholipid, although a slight time delay

was often seen. Phagolysosomes isolated at 0, 2, 5, and 24 hours after phago-

-I 0 2 4 6 8 IO Hours

FIG. 3. Resumption of pinocytosis and phagocytosis. . . _ ̂ . Macro- phages on coverslips were exposed to 1 mg of polystyrene latex particles, l.l-pm diameter per ml of 20% NBCS for 1 hour, washed free of extracellular beads, then assayed for endocytic activity at various times after ingestion. For assaying pinocytic activity, macrophages were placed in 507, NBCS after phagocytosis. The coverslips were fixed in 1.257, glutaraldehyde and the number of pinocytic vesicles (P.V.) counted as described previously (28), and expressed as percentage seen in control cells which had not phagocytized latex particles (0). At the beginning of the experiment the controls had 425 P.V. per 50 cells, and 485 P.V. per 50

cells at 24 hours. For assaying phagocytic activity, macrophages were placed in 20% NBCS. At the assay times, the cells were placed in 20% NBCS containing 5 X lo1 1.96-pm latex beads per ml for 30 min at 37”, then fixed in glutaraldehyde. The number of test particles ingested was compared to the number ingested by control cultures (0). The controls contained 457 particles per 100 cells.

cytosis contained decreasing amount of 5’-nucleotidase (Table V). By 5 hours after phagocytosis 5’-nucleotidase specific activity was less than half of the homogenate value. The homog-

enate specific activity also decreased during the first 5 hours after ingestion. By 24 hours the homogenate specific activity had returned to control levels; however, the specific activity in


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2444 Membrane Xynthesis in Macrophages after Phagocytosis

Cholesterol Phospholipld

0 2 4 6 8 IO Hours after Phagocytosis

5’Nucleotidase

I

Vol. 247, No. 8

FIG. 4. Effect of phagocytosis of latex particles on cholesterol, phospholipid, and 5’-nucleotidase content of macrophages. Macro- phage flask cultures were incubated in 209$ NBCS containing 0,250,500, and 1OOOrg of 1.1~pm latex particles per ml for 1 hour, washed, then incubated in fresh 20% NBCS. After ohaeocvtosis the macrophages contained 0 (0), 14 f 4 (X), 31 f 8 (a), and 47 f 7 (0) pg of latex per flask, respectively.

_ Y”

TABLE V

Change in 5’-nucleotidase content of phagolysosomes after phagocytosis

Latex phagolysosomes (lo/20 IF fractions) were prepared from macrophages incubated in 20% NBCS at various times after phagocytosis. The recovery of 5’-nucleotidase in phagolysosomes was corrected to account for latex found in other fractions. The relative specific activity is compared to the homogenate. The activity of the homogenate is in specific activity @.A.) units as in Table I. Phagolysosomes isolated at 24 hours after phagocytosis, and at 24 hours after phagocytosis in the presence of 2 mM NaF for 4 hours nrior to fractionation, designated as time 24 (NaF).

Time after phagocytosis

hrs

0 2 5

24 24 (NaF)

Specific activity >f 5’.nucleotidas

in homogenate

93 82 53 86

103

5’.Nucleotidase in phagolysosomes

Recovery of homogenate

percentage Relative S.A.

%

34 2.5 23 1.5

8 0.35 2 0.15

<l <0.05

the phagolysosomes was less than 20% of the homogenate value. If, before isolation of the phagolysosomes, the macrophages were incubated in serum medium containing 2 mM sodium fluoride, an inhibitor of pinocytosis and phagocytosis (27), then the 5’.nucleotidase activity of the latex phagolysosomes decreased to trace levels.

Effect of Exogenous Cholesterol on Increase of Macrophage Cholesterol and 5’Wucleotidase after Phagocytosis

In the absence of exogenous cholesterol macrophages do not synthesize cholesterol from [Wlacetate. The influence of a cholesterol-depleted medium on the synthesis of membrane following phagocytosis was next examined. As seen in Fig. 5, cultivation in bovine serum albumin medium following latex ingestion blocked the increases in cell cholesterol. 5’-Nucleo- tidase activity decreased to a minimum value at 6 hours, but did not recover to control values by 10 hours. Phospholipid

4.0 Cholesterol 5JNucleot~dase

0 2 4 6 8 IO Hours after Phaqocytosls

FIG. 5. Effect of exogenous cholesterol on changes in cellular cholesterol and 5’nucleotidase after phagocytosis. After phagocytosis, macrophages were incubated in 20% NBCS (X) or in cholesterol-free 10 mg per ml of bovine serum albumin in minimal Eagle’s medium (0). Each culture contained 52 f 14 pg of latex. Nonphagocytizing cultures (a) were incubated in 20% NBCS.

phosphorus also did not increase in the cultures incubated in bovine serum albumin.

Effect of Inhibition of Protein Synthesis

The effects of puromycin at a level which blocks >SO% of macrophage protein synthesis are shown in Fig. 6. Addition of the inhibitor at 0 and 4 hours after phagocytosis blocked the subsequent increase in cell cholesterol and 5’nucleotidase activity. Addition of the inhibitor 6 hours after ingestion did not affect the net increment in cholesterol levels. In contrast, 5’- nucleotidase recovery to control levels failed to occur when macrophages were exposed starting at 6 hours after ingestion. Cell viability remained at >95% during the experiment. Both total and specific activities decreased in the puromycin-treated cells. Controls which had not ingested latex also decreased somewhat in 5’nucleotidase activity during a continuous IO-hour exposure to puromycin.

Effect of Inhibition of RNA Synthesis

Bromotubercidin was used to inhibit RNA synthesis in macrophages (29) after ingestion of latex (Fig. 7). A continuous exposure for 10 hours starting immediately after phagocytosis


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Cholesterol 40

1

Puromycln

I 5’Nucleotidase [

Hours after Phagocytosis

FIG. 6. Effect of protein synthesis inhibition on cholesterol an d 5’-nucleotidase levels after phagocytosis. Macrophages were incubated in 20y0 NBCS. Puromycin (5 pg per ml) was added to the cultures for periods 0 to 10 hours (A), 4 to 10 hours (m), 6 to 10 hours (X), or no puromycin (0). Nonphagocytizing controls received no puromycin (0)) or puromycin (5 pg per ml) at 0 to 10 hours (A), or 4 to 10 hours (0).

40. Cholesterol I 5’Nucieotldase 1

0 2 4 6 8 IO Hours after Phagocytosls

FIG. 7. Effect of RNA synthesis inhibition on cholesterol and 5’-nucleotidase levels after phagocytosis. Macrophages were incubated in 20% NBCS. Bromotubercidin (5 pg per ml) was added to the cultures in the same experiment as Fig. 6 for periods of 0 to 10 hours (A), 4 to 10 hours (m), 6 to 10 hours (X), or no bromotubercidin (0). Nonphagocytizing controls received no inhibitor (O), or bromotubercidin (5 rg per ml) at 0 to 10 hours (A), or 4 to 10 hours (0).

inhibited the increase in cholesterol content of the cells. Addi- tion of the inhibitor at 4 hours or later had no significant effect on the cholesterol changes. Phospholipid increases were affected in a similar manner. Cells exposed to bromotubercidin for 10 hours after ingestion exhibited the usual reduction in 5’-nucleo-

tidase activity, but this activity did not return to control levels at 10 hours. Exposure to the drug starting at 4 hours also inhibited the recovery of 5’.nucleotidase activity, but was less pronounced than for the IO-hour exposure.

DISCUSSION

Morphological observations indicate that the membrane of the phagocytic vacuole is derived from the plasma membrane.

The lysosomal membrane is similar to the plasma membrane in lipid composition (30), glycoprotein-staining characteristics (31), and they share common antigens (32). We have reported here the transient presence of 5’.nucleotidase, a plasma membrane marker enzyme (7), on the membrane of phagolysosomes. This enzyme is identical with the plasma membrane enzyme with criteria of substrate specificity, pH optimum, and zinc ion inhibition, and are similar to 5’-nucleotidases of other tissues (21). The amount of enzyme found in the phagolysosomes is proportional to the amount of plasma membrane interiorized by ingestion of latex particles, and to the increase in cholesterol attributable to the increase in lysosomal membrane immediately after phagocytosis (2). The enzyme has an intralysosomal half-life of about 2 hours. Its disappearance may represent modification of the membrane after interiorization, perhaps by hydrolysis, or removal from the membrane, or steric changes. Macrophages also have on their surface a divalent cation requir- ing adenosine triphosphatase. Following membrane interiorization, the cytochemically demonstrable activity on pinocytic vacuoles is rapidly lost (33). This suggests that the membrane of the pinocytic vesicle is also modified some time after separa- tion from the surface. Indeed, pinocytic activity appears to be the source of the low levels of 5’.nucleotidase found in phagolysosomes isolated 24 hours after phagocytosis.

Prior to ingestion of latex, the plasma membrane accounts for essentially all of the cellular 5’-nucleotidase. After phagocytosis, a fraction of the enzyme proportional to the amount of membrane interiorized is found in the phagolysosomes. This activity decreases while the remaining activity attributable to the plasma membrane is not altered. This results in a decrease in total enzyme activity which reaches a minimum at about 6 hours. Then the enzyme activity begins to increase approaching control levels by 10 to 12 hours. This activity is not associated with phagolysosomes. Since RNA and protein synthesis are required, and a time delay is involved, it is unlikely that the enzyme removed from the lysosomal membrane is reutilized in the plasma membrane.

Parallel increases in cholesterol and phospholipid indicative of cholesterol-rich cytomembranes occur after phagocytosis in conjunction with the increase in 5’-nucleotidase. The net increase is clearly related to the amount of plasma membrane initially interiorized. Previous studies on the localization of ex- changeable cholesterol pools in macrophages indicated that plasma membrane cholesterol reached control levels by 12 hours after phagocytosis, while the net increase in lysosomal membrane cholesterol occurred immediately after ingestion (2). This indicated that the net increment in cholesterol and phospholipid was to restore the plasma membrane to its original size although a small contribution of other cholesterol-containing membranes such as Golgi membranes cannot be excluded. A net increase in lysosomes at this time is unl.kely, as indicated by the cholesterol pool analysis (2), and the lack of changes in acid hydrolase levels after phagocytosis of latex (34). Since plasma membrane functions such as pinocytosis and phagocytosis resume in parallel to the increases in membrane constituents, it may be con- cluded that plasma membrane synthesis occurs at this time.

Among the membrane proteins which need to be replaced is a trypsin-labile site which acts as a receptor for serum lipoproteins which are the source of membrane cholesterol (2). Regenera- tion of this receptor after trypsinization takes 7 hours, which is similar to the time delay for membrane synthesis after phagocy-


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tosis. 5’-Nucleotidase activity appears to be restored to the membrane slightly later than the structural components, as shown by the differential recovery during RNA and protein synthesis inhibition. Thus a 5’-nucleotidase-deficient membrane may be formed. However, in the absence of the exogenous cholesterol required for the membrane, increases in phospholipid and 5’-nucleotidase do not take place, although the cells remain viable. This structural component may control the assembly of the membrane from newly synthesized and previously existing constituents. In a fatty acid auxotroph of Escherichia coli, the membrane-associated lactose permease is not induced in the absence of the fatty acid required for phospholipid synthesis (35). Since phospholipids may be associated with cholesterol in a constant stoichiometric relationship (36), control of membrane assembly may occur at this level in the macrophage. In the case of 5’.nucleotidase, which is associated with sphingo- myelin (II), the failure to sy-nthesize phospholipids may be the controlling factor. The control of the amount of new plasma membrane is clearly related to the amount of membrane interiorized, but the mechanism of this feedback control is unknown.

Pinocytosis of nondigestible molecules also results in increased numbers of secondary lysosomes, which maintain a constant size (2). Since accumulation of the storage granules occurs over a prolonged period of time, net membrane synthesis prob- ably occurs continually and thus would be difficult to show. If digestible substances are taken up by endocytosis, a stable population of lysosomes is not formed (34, 37), and it is unknown what effect this might have on membrane synthesis.

Earlier studies of membrane events related to phagocytosis have dealt with the increased turnover of phospholipids during phagocytosis in polymorphonuclear leukocytes and macrophages (3-6). The increased labeling of the phospholipids was often small, and no net accumulation of phospholipid resulted. The assumption made by these studies was that membrane synthesis was involved in the formation of the phagocytic vacuole and occurred during particle ingestion. We have reported here that demonstrable membrane synthesis occurs about 6 hours after ingestion of polystyrene latex particles. Increases as large as 100% in cellular cholesterol and phospholipid take place. The phenomenon of increased turnover of lipids during phagocytosis may be quite different in function from the net synthesis of membrane after phagocytosis. The former may represent alterations of the plasma membrane to form the membrane of the phagocytic vacuole, or may be related to the changes in metabo- lism during phagocytosis. It may be significant that the increased turnover occurs primarily in polymorphonuclear leukocytes which are short lived, end cells containing a nonrenewable population of granules (38). Synthesis and recycling of membrane may have little importance in the functions of this cell. In contrast, the macrophage is long lived and is capable of syn- thesizing new primary lysosomes and lysosomal enzymes (8).

Since substances not degraded by lysosomal hydrolases remain within lysosomes indefinitely (34, 37, 39), net. synthesis of membrane would be required to maintain the funct,ional integrity of the cell surface.

1. 2. 3. 4. 5.

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EMMELOT, P., Bos, C. J., BENEDETTI, E. L., AND R~JMKE, P. (1964) Biochim. Biophys. Acta 90, 126-145

COHN, Z. A., AND BENSON, B. (1965) J. Exp. Med. 121, 153-169 WETZEL, M. G., AND KERN, E. D. (1969) J. Cell Biol. 43, 90 WARREN, L., CLICK, M. C., AND NASS, M. K. (1966) J. Cell.

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Arch. Biochem. Biophys. 56, 165 HEPPGL, L. A., AND HILMOE, R. J. (1951) J. BioZ. Chem. 188,665 COHN, Z. A. (1968) Advan. Zmmunol. 9, 163 CHAPMAN-ANDERSEN, C. (1962) C. R. Trav. Lab. Carlsberg 33,

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REFERENCES


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Zena Werb and Zanvil A. CohnPolystyrene Latex Particles

Plasma Membrane Synthesis in the Macrophage following Phagocytosis of

1972, 247:2439-2446.J. Biol. Chem.

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