phagocytosis by macrophages - journal of cell science

J. Cell Sci. 51, 189- aoi (1981)Printed in Great Britain © Company of Biologists Limited 1981

PHAGOCYTOSIS BY MACROPHAGES

II. THE DISSOCIATION OF THE ATTACHMENTAND INGESTION STEPS

TADANAO ITO, MASAMICHI J. UEDA*. T. S. OKADAAND SHUN-ICHI OHNISHIDepartment of Biophysics, Faculty of Science, Kyoto University,Kyoto, Japan

SUMMARY

The phagocytic process of mouse peritoneal macrophages was dissociated, using bovineserum albumin (BSA)-coated particles containing spin-labelled cholestanone, into 2 steps:attachment of particles to the cell surface and ingestion of the particles into the cytoplasm.The number of particles was estimated from electron spin resonance (e.s.r.) measurements.The particles ingested into the cytoplasm were distinguished from those attached to thecell surface by treatment with a membrane-impermeable reducing agent, ascorbate. Thevalidity of the assay method was tested under various conditions. The measurements piovidedaccurate and reproducible data. The phagocytic reaction was followed as a function of timeand the rate constants for the attachment and ingestion steps were obtained from the initialphase. Both steps were highly dependent on temperature. Divalent cations in the incubationmedium were essential for the attachment step but apparently had no effect on the ingestionstep. The metabolic inhibitors, KCN and 2-deoxyglucose, inhibited both steps. Cytochalasin Binhibited both steps, while colchicine inhibited only the attachment step but apparently hadno effect on the ingestion step.

INTRODUCTION

Phagocytosis is a fundamental cellular function that internalizes exogenous particlesinto the cytoplasm (Silverstein, Steinman & Cohn, 1977). After the attachment ofparticles to the cell surface, pseudopodia appear and extend to embrace the particulateobject. The membrane pleats surrounding the particle ultimately fuse and encasethe object within a phagocytic vesicle. The involvement of cytoplasmic contractileproteins in the extension of pseudopodia and the engulfment of particles has beensuggested (Stossel & Hartwig, 1976).

The phagocytic process can be divided experimentally into 2 discrete steps:attachment and ingestion of particles. The method used was direct microscopiccounting of the number of particles attached to the cell surface and internalizedinto the cell (Allen & Cook, 1970; Michl, Ohlbaum & Silverstein, 1976; Rabinovitch,1967). Since the number of cells counted is limited for technical reasons, themeasurements are not accurate enough for a kinetic analysis of phagocytosis. A moreaccurate but indirect method has also been used to quantitate the number of particles.

• Present address: Department of Pathology, Institute for Virus Research, Kyoto University,Kyoto, Japan.

190 T. Ito, M. J. Veda, T. S. Okada and S. Ohnishi

After being freed from unattached particles, the number of ' phagocytized' particleswas measured by spectrophotometry or by counting radioactivity (Weisman & Korn,1967; Michell, Pancake, Noseworthy & Karnousky, 1969; Stossel, Mason, Hartwig &Vaughan, 1972; Stossel, 1973). Analysis of the phagocytic reaction in terms of theMichaelis-Menten type of mechanism has been carried out based on these data.However, this method cannot be used to discriminate between the attached andingested particles.

We have developed a new spectrophotometric method that can dissociate thephagocytic reaction into attachment and ingestion steps. The method employselectron spin resonance (e.s.r.) measurement of spin-labelled cholestanone dissolvedinto bovine serum albumin (BSA)-coated paraffin particles. The intensity of thesignal of cell-associated particles gives a measure of the sum of the attached andingested particles, and the intensity after treatment of the cells with ascorbateprovides a measure of the ingested particles, since ascorbate reduces the spin labeland destroys the e.s.r. signal, and because the cell membrane is practically imper-meable to ascorbate at low temperatures. After experimental tests of the validityof the method, we have investigated the effect of temperature and various additiveson the 2 individual steps of phagocytosis of mouse peritoneal macrophages.

MATERIALS AND METHODS

Macrophages

The cells were harvested from the peritoneum of female 3-month-old mice, strain ddY,which had been injected intraperitoneally with 2 ml of thioglycolate medium 4 days before(Ichikawa, Pluzik & Sachs, 1967). The cells were washed 3 times with Hanks' saline containingo-1 % (w/w) glucose buffered at pH 74 with 10 mM-.N-2-hydroxyethylpiperazine-2Vv-2-ethane-sulphonic acid (Hanks/HEPES/glucose).

Preparation of particles for phagocytosis

An emulsion of paraffin oil coated with BSA or opsonized with complement was preparedby modification of the methods of Stossel et al. (1972) and Stossel (1973) as described below.

BSA-coated particles. One ml of paraffin oil (Merck) containing cholestanone spin label(10 mg/ml) was layered on 3 ml of Hanks/HEPES/glucose containing 2 % (w/w) BSA (fractionV, Reheis Chemical Co.). The 5 mm-0 tip of a 20 kHz sonicator (Kaijo Denki Co.) wasplaced above the oil-water interface and emulsion was achieved at 250 mA of plate currentfor 10 min at o °C. The density of the emulsion was about 088 g/cm' and all the cholestanonespin label remained in the emulsion. The spin label, a 7V-oxy-4',4'-dimethyloxazolidinederivative of cholestanone, was synthesized according to Keana, Keana & Beetham (1967).Unless indicated otherwise, BSA-coated particles were used in the experiments.

Complement-opsonized particles. One ml of paraffin oil containing 10 mg/ml cholestanonespin label was emulsified in 3 ml of Hanks/HEPES/glucose containing 60 mg lipopolysaccharide(Escherichia colt 0127 :B,; Difco) by the method described above. The paraffin oil particleswere washed twice before opsonization. The washing was accomplished by centrifugation at10000 g for 10 min, removal of the infranatant fluid and resuspension of the particles inHanks/HEPES/glucose. After washing, the particles were suspended in 3 ml of Hanks/HEPES/glucose. In order to opsonize the particles with complement, 3 ml of fresh mouse serum(ddY?) was incubated with an equal volume of particles for 15 min at 37 °C. The opsonizedparticles were washed as described above and suspended in 3 ml of Hanks/HEPES/glucose.

Phagocytosis by macrophages 191

Assay of attachment and ingestion of particles

Cells suspended in 1 ml of Hanks/HEPES/glucose (~ io7 cells/ml) were preincubated ina test tube at 37 °C for 10 min and 0-25 ml of the paraffin emulsion (prewarmed at 37 °C)was added and the suspension was incubated for the appropriate time. At the end of theincubation, 6 ml of iV-ethylmaleimide (1 min) in 145 mM-NaCl was added to the cellsuspension.

For the assay of total associated particles, the cell suspension was centrifuged at 1500 g for5 min at 4 °C and the supernatant was discarded. The inside of the test tube was wiped withtissue paper to remove the particles remaining on the surface. The pellet was placed in aquartz capillary tube and the e.s.r. spectrum was measured at 22 °C with a JEOLCO modelME-X spectrometer. The central peak height was read and used to quantitate the particlesattached and ingested inside the cells.

For the assay of ingested particles, the cell suspension was centrifuged at 400 g for 5 minat 4 °C and the supernatant was discarded. The inside of the test tube was wiped as mentionedabove. One ml of ascorbate solution was added to the pellet and it was kept for 90 min ato °C. The ascorbate solution consisted of 07 ml of Hanks/HEPES/glucose containing 1 -4 mM-iy-ethylmaleimide and 0-3 ml of freshly prepared 300 mM-sodium ascorbate (pH adjusted to7-4). The mixture was then centrifuged at 600 g for 5 min at 4 °C. The pellet was taken intothe capillary and the e.s.r. spectrum was measured to quantitate the ingested particles.

RESULTS

Rationale of the method of assay

The method utilizes the reduction of the spin label nitroxide radical by ascorbateand the almost complete impermeability of the cell membrane to ascorbate at lowtemperatures. Fig. 1, curve A shows the decay of the e.s.r. signal of cholestanonespin label contained in the BSA-coated paraffin particles when treated with ascorbateat o °C. The intensity gradually decreased and became less than 5 % of the initialvalue after 90 min. On the other hand, when the particles were incubated withmacrophages at 37 °C and the cells were treated with ascorbate at o °C, the signalintensity decreased but to a limited extent and did not decay further (Fig. 1, curve B).A control experiment without addition of ascorbate showed no decay of the signalin the time interval. The results therefore indicate that about 50% of the particleswere located on the outside of the cells where ascorbate was accessible, and therest of the particles were ingested inside the cells and protected from reduction byascorbate. It is also shown that the spin label in the ingested particles was protectedfrom various reducing agents in the cytoplasm.

The above conclusion was confirmed by another experiment in which complement-opsonized paraffin particles were used. After incubating macrophages with opsonizedparticles at o °C for 60 min, unattached particles were removed by centrifugation.The cell pellet was resuspended in 2 ml of Hanks/HEPES/glucose and incubatedfor the appropriate time at 37 °C. Ascorbate was added to the cells and they werekept for 90 min at o °C. The remaining e.s.r. signal was larger for the cells incubatedfor a longer time at 37 °C. After incubation for 30 min, the e.s.r. signal intensitybecame almost the same as that of cells not treated with ascorbate (Fig. 2, curve B).The signal intensity of ascorbate-untreated cells did not decrease during the incubation(Fig. 2, curve A). These results show that, during the incubation, the attached

I 9 2 T. Ito, M. J. Ueda, T. 3. Okada and S. Ohnishi

30 60 90

Treatment time (min)Fig. i. Reduction of cholestanone spin label in BSA-coated paraffin particles byascorbate. Curve A, particles only; ascorbate was added at o CC to the particlesuspension and the e.s.r. signal was measured at given times. Curve B, after incubationwith macrophages; the cells were incubated with the particles at 37 °C for 4 minand centrifuged. Ascorbate was added at o °C to the cell pellet and the e.s.r. signalwas measured at the times given in the text.

» 0-6 -

10 20 30Incubation time (min)

Fig. 2. Increase in the number of ingested particles with incubation. Macrophageswere incubated with the complement-opsonized particles at o CC for 60 min andcentrifuged. The cells were resuspended in Hanks/HEPES/glucose, incubated at37 °C for various periods, and the e.s.r. spectrum was measured before treatmentwith ascorbate {A) or after (B). Ascorbate was added to the cells (at o °C), whichwere kept for 90 min before the e.s.r. measurement. The signal intensity is plottedagainst incubation time at 37 °C.


particles were ingested into the cytoplasm and completely protected against ascorbateand endogenous reducing agents in the macrophages.

The treatment with ascorbate did not affect cell viability. The cells treated withascorbate for 90 min at o °C were cultured overnight in a Falcon plastic dish con-taining Eagle's minimal essential medium supplemented with 6 % foetal calf serumunder a humid atmosphere of 95 % air and 5 % CO2 at 37 °C. Almost all the macro-phages were intact and adhered to the dish. These cells were also found to be viableby the trypan blue dye exclusion test (Boyes, Old & Charoulikov, 1964).

Kinetic analysis of phagocytosis

In this study, the phagocytic process was treated as 2 sequential steps: irreversibleattachment and ingestion.

V v

Particles in the medium > particles attached to the cell surface^) >•particles ingested inside the cell (n2).

V represents the rate of irreversible attachment and v the rate of ingestion.The e.s.r. signal intensity of the cells after incubation with spin-labelled paraffin

particles gives the total number of attached and ingested particles i^ + n^), and thatof the cells after treatment with ascorbate gives the number of ingested particles(n2). The following equations hold, where t is time:

dnjdt = V-v, (1)

dn2/d* = v. (2)

From equations (1) and (2)

d(n1 + n2)/d* = V (3)

follows. When (nx + n^) was plotted against incubation time at 37 °C, the curveinitially followed a straight line and then gradually deviated (Fig. 3). Therefore, theslope of the linear region corresponds to the initial rate of irreversible attachment(J9. Within this time interval (4 min at 37 °C),

nx + n2 = V,t (4)

can hold.

We represent the rate of ingestion, v, as

v = kjilt (5)

where k2 corresponds to the rate constant of ingestion.From equations (2), (4) and (5), the following solution is obtained:

«2 = K[* + ( e - * ' t - i ) / * J . (6)

The validity of this kinetic treatment is demonstrated in Fig. 4, in which theexperimental values of n2 are plotted together with the theoretical curve calculatedusing equation (6) and k^ = 0-27 min"1.

The fraction of particles ingested, P, is given by

^ . (7)

194 T. ho, M. J. Veda, T. S. Okada and S. Ohnishi

1-5 -

2 4 6 8Incubation time (min)

Fig. 3. Time course of the increase in the number of cell-associated particles withincubation. Macrophages were incubated with the BSA-coated particles at 37 °Cfor the indicated times and centrifuged. The e.s.r. signal intensity of the cell pelletis plotted against incubation time.

10

g 0-5

I I I

2 4 6Incubation time (min)

Fig. 4. Comparison of experimental number of ingested particles (O) with theoreticalcurve ( ), calculated using equation (5) with kt = 0-27 min-1. The value at 4 minwas fitted to the theoretical curve. The ordinate is the e.s.r. signal intensity aftertreatment with ascorbate of macrophages that had been incubated with the BSA-coated particles at 37 °C for the times indicated.


The fraction P is thus dependent only on k2, whereas the total number of associatedparticles depends only on T̂ .

Kinetic analysis of the initial rate, Vl} has been performed by Wiesman & Korn(1967), Stossel et al. (1972), and the present authors (Ueda, Ito, Ohnishi & Okada,1981) postulated an intermediate step of reversible attachment. The main interestin the present study is to discriminate between irreversible attachment and ingestionand therefore such a kinetic analysis was not done.

In routine assays, the phagocytic reaction was carried out for 4 min at 37 °C, andthe total number of associated particles and the fraction of ingested particles were

0-5 -

0-3

0-1

- 100

I

50

10 4020 30Incubation temperature (°C)

Fig. 5. Temperature dependence of the attachment and ingestion steps of phago-cytosis. The cells were incubated with the BSA-coated particles for 4 min at theindicated temperatures and values of (f̂ + Wi) ( • ) and P(O) were obtained fromthe e.s.r. measurements, (n^ + n^) is given as a percentage of the value at 37 CC.

obtained from the e.s.r. signal intensity. Average values of {n^ + n^) and P wereabout 0-5 mg paraffin emulsion per io7 cells and 0-4, respectively. The absolutevalues varied with different batches of cells prepared separately. This appears to bedue mainly to differences of cell viability. A series of experiments was carried outon cells derived from the same batch. The reproducibility of data was quite good towithin 95%. The effects of drugs, divalent cations and temperature were expressedas the ratio to the control («i + «2)

w a s calculated to measure the attachment stepand P the ingestion step, according to equations (4) and (7).

Effect of temperature

Fig. 5 shows effect of the incubation temperature on the attachment and ingestionsteps of the phagocytic reaction. Both (nx + «8) and P decreased markedly withdecreases in temperature. For example, the ratio of (nl + n2) at 25 °C to that at


37 °C was 0-19 and the ratio of P at 25 °C to that 37 °C was 0-34. The activationenergy of the ingestion rate, kit was determined to be 22 kcal moh1 from equation (7).

Effect of metabolic inhibitors

Since phagocytosis is known to be an energy-dependent process, the effects ofKCN, an inhibitor of oxidative metabolism and 2-deoxyglucose, a glycolytic inhibitor,were investigated. The results showed that these metabolic inhibitors inhibitedboth the attachment and ingestion steps (Fig. 6). Addition of 1 mM-KCN decreased

to 48% and P to 60% of the control; 2-deoxyglucose at 5 nffl decreased

A KCN

100 -

n,+ n2 P

( ) B 2-Deoxyglucose

5 0 -

0-2 -

I

0-3

0-2

100-

I001 0-5 10 5 0 10 50

Concentration (nriM) Concentration

Fig. 6. Effect of metabolic inhibitors on the attachment and ingestion steps ofphagocytosis. Macrophages were preincubated at 37 CC for 10 min in Hanks/HEPES/glucose containing the indicated concentrations of KCN (A) or 2-deoxyglucose (B)and then incubation with the BSA-coated particles for 4 min at 37 °C. ( • ) (rtx + nj;(O)P.

(nl + ni) to 24% and P to 63% of the control. Glucose added to 5 mM did not affectthe phagocytic reaction. These results indicates that the rates of irreversible attach-ment and ingestion were decreased to 48 and 46% by 1 mM-KCN and to 24 and47 % by 5 mM-2-deoxyglucose, respectively.

Effect of divalent cations

In order to investigate the effect of divalent cations, the cells were preincubatedat 37 °C in Hanks/HEPES/glucose containing only the indicated cation, and thenincubated with BSA-coated paraffin particles. The results are summarized in Table 1,which shows, firstly, that the divalent cations are indispensable for the attachmentstep. Omission of divalent cations at various concentrations markedly reduced the


Table i. Effect of divalent cations on attachment andingestion steps of phagocytosis

Divalent cation

Mg1+

Ca«+

Mn'+

Co«+

Concentration(mM)

i - o

O-2

i - o

0-05o-oz0 - 2

0-05

(Vr + n,)(». + ",),

0 6 30-36

o-340 7 1

0-620-690-59

pPo

I - I

I - I

I - I

I - I

I-O

I - I

0-4 0-70 i-oO-I5 0-44 I-I

Hanks/HEPES/glucose normally contains i-2mM-Mg*+ and o-8 mM-Ca*+. The mediumwas depleted of these divalent cations and the appropriate cation was added to the medium asrequired. Macrophages were preincubated in the medium and mixed with the spin-labelledparaffin particles that had been emulsified in divalent-free medium. The BSA coated on theparticles was dialysed on divalent-free medium before use. Incubation was for 4 min at37 °C (nj +«,) and P values are given as the ratios to the control (i.e. in 1 -2 mM-Mg'+ ando-8 mM-Cal+). The subscript c means control.

0-8

0-6

0-4

5 10Incubation time (mm)

Fig. 7. Effect of divalent cations on the increase in the number of ingested particleswith incubation. Macrophages were incubated with the BSA-coated particles at37 °C for 4 min and centrifuged. The cells were resuspended in Hanks/HEPES/glucose containing 1-2 mM-Mgt+ and o-8 mM-Ca1+ ( • ) , or 0-5 mM-EDTA withoutdivalent cations (O). and incubated at 37 °C for various periods of time. Ascorbatewas added to the cells at o °C, and after 90 min the e.s.r. spectrum was measured.The signal intensity is plotted against incubation time at 37 °C.


0-4- -

0-2 -

1 0

- 100

+

10

Concentration

- 100

+c

10

Concentration (J/M)Fig. 8. Effect of cytochalasin B (A) and colchicine (B) on the attachment and ingestionsteps of phagocytosis. Macrophages were preincubated in Hanks/HEPES/glucosecontaining the indicated concentrations of drugs at 37 °C for 10 min and thenincubated with the BSA-coated particles at 37 °C for 4 min. (#) («i + n,); (O) P-

number of associated particles. The divalent cations varied in effectiveness. Secondly,and more interestingly, the divalent cations did not apparently affect the ingestionstep. P/Pc remained at i-o to 11.

This conclusion was ascertained by more-direct experiments. After incubationwith the BSA-coated particles at 37 °C for 4 min, the cells were collected by centri-fugation and resuspended in Hanks/HEPES/glucose containing 05 mM-EDTA butno divalent cations. In a control experiment, the cells were resuspended in normalHanks/HEPES/glucose that contained 12 mM-Mg2+ and o-8 mM-Ca2+. The cellsuspension was incubated at 37 °C for the appropriate time, centrifuged, and treatedwith ascorbate. The remaining e.s.r. signal increased with incubation time. It wasremarkable that the time course for the cells incubated in the absence of divalent


cations could be superimposed on that of the control as shown in Fig. 7. This resultstrongly indicates that the divalent cations had no effect on the ingestion step.

Effect of cytochalasin B and colchicine

In order to examine the involvement of cytoplasmic fibrous proteins in the stepsof phagocytosis, the effects of the drugs on the 2 different steps were investigated.Cytochalasin B markedly inhibited both steps but colchicine did not affect the ingestionstep (Fig. 8). Colchicine inhibited the attachment step but relatively weakly.

DISCUSSION

Possible steps in the whole process of phagocytosis are presented in Fig. 9. Theseinclude reversible (a) and irreversible (b) attachment of particles, extension ofpseudopodia (c), and membrane fusion and ingestion (d). The stage at whichascorbate cannot attack the particles is E. In the present analysis, the overall stepfrom A to C was treated as attachment and the sequential steps from C to E as

<4

E D

Fig. 9. Possible steps in phagocytosis, (a) Reversible attachment; (6) irreversibleattachment of a particle, (c) Membrane envelopment around the particle, (d) Mem-brane fusion and ingestion of the particles into the cytoplasm. Stages A to E aredescribed in the text.

ingestion. The overall step from A to C could be analysed including an intermediatestate, B, as done by Weisman & Korn (1967), Stossel (1973) and also by the presentauthors (Ueda, Ito, Ohnishi & Okada, 1981). However, our main interest in the presentanalysis is to discriminate between the irreversible attachment and ingestion steps,which cannot be done by the Michaelis-Menten type of analysis. The ingestion rate,&2, is the sum of the rates of the 2 processes, (c) and (d), i.e. membrane envelop-ment and fusion.

The results suggest that some rearrangements of cell-surface receptors are involvedin the irreversible attachment step, since lowering the temperature, addition ofmetabolic inhibitors, and cytochalasin B inhibited the step. The ingestion stepconsists of extension of pseudopodia and membrane fusion around the particle. It is

200 T. Ito, M. J. Ueda, T. S. Okada and S. Ohnishi

well known that cytoplasmic contractile proteins such as actin have an importantrole in the extension of pseudopodia (Stossel & Hartwig, 1976). The inhibition ofthe ingestion step by cytochalasin B and metabolic inhibitors is therefore quiteunderstandable because they impair the function of cytoplasmic contractile proteins.

The role of divalent cations is unique since they were indispensable for irreversibleattachment but had no effect on the ingestion step. When the cell-particle complexwas brought to the irreversible attachment state (C) with the help of divalent cations,the following steps in the process did not require those cations, suggesting thatextension of pseudopodia and membrane fusion will occur without external divalentcations. Stimulation of the rate of particle uptake by divalent cations has also beenindicated from Michaelis-Menten type analysis of the phagocytic reaction of granu-locytes and rabbit alveolar macrophages (Stossel, 1973). The present analysis givesfurther insight into the role of divalent cations.

Rabinovitch (1967) has dissociated the steps of attachment and ingestion in mouseperitoneal macrophages by experiments using optical microscopy. Both steps weredependent on temperature, in qualitative agreement with our results. However, therequirement of divalent cations in the 2 individual steps was the opposite to thefindings of the present results. Michell et al. (1969) have observed that 2-deoxyglucosehad no inhibitory effect on the capacity of mouse peritoneal macrophages to phagocytizelatex or zymosan particles. The discrepancy with our results may be due to differentexperimental conditions. These authors observed phagocytosis when the uptake ofparticles proceeded virtually to completion, while we measured the initial rate.

The spin-label assay can provide accurate data on the 2 individual steps in phago-cytosis. The errors in a series of experiments were well within 5%. The accuracy,perhaps, is due to the fact that the measurements made were the average valuesfrom ~ io7 cells, in comparison with ~ io2 cells in the microscopic method. Detailedinformation on individual steps would facilitate our understanding of the wholephagocytic reaction in molecular terms.

REFERENCES

ALLEN, J. M. & COOK, G. M. W. (1970). A study of the attachment phase of phagocytosis bymurine macrophages. Expl Cell Res. 59, 105-116.

BOYES, E. A., OLD, L. J. & CHAROULIKOV, J. (1964). Cytotoxicity test for demonstration ofantibodies. Meth. med. Res. 10, 39—47.

ICHIKAWA, Y., PLUZIK, D. H. & SACHS, L. (1967). Feedback inhibition of the developementof macrophage and granulocyte colonies. I. Inhibition by macrophage. Proc. natn. Acad.Set. U.S.A. 58, 1480-1486.

KEANA, J. F. W., KEANA, S. B. & BEETHAM, D. (1967). A new versatile ketone spin label.J. Am. chem. Soc. 89, 3055-3056.

MICHELL, R. H., PANCAKE, S. J., NOSEWORTHY, J. & KARNOVSKY, M. L. (1969). Measurementof rats of phagocytosis. The use of cellular monolayers. J. Cell Biol. 40, 216-224.

MICHL, J., OHLBAUM, D. J. & SILVERSTEIN, S. C. (1976). 2-Deoxyglucose selectively inhibitsFc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages.I. Description of the inhibitory effect. .7. exp. Med. 14, 1465-1483.

RABINOVITCH, M. (1967). The dissociation of the attachment and ingestion phases of phago-cytosis by macrophages. Expl Cell Res. 46, 19-28.

Phagocytosis by tnacrophages 201

SILVERSTEIN, S. C , STEINMAN, R. M. & COHN, Z. A. (1977). Endocytosis. A. Rev. Biochem.46, 669-722.

STOSSEL, T. P. (1973). Quantitative studies of phagocytosis: Kinetic effects of cation andheat-labile opsonin. J. Cell Biol. 58, 346-356.

STOSSEL, T. P. & HARTWIG, J. H. (1976). Phagocytosis and the contractile proteins of pulmonarymacrophages. In Cell Motility, book B (ed. R. Goldman, T. Pollard & J. Rosenbaum),pp. 529-544. New York: Cold Spring Harbor Laboratory.

STOSSEL, T. P., MASON, R. J., HARTWIG, J. & VAUGHAN, M. (1972). Quantitative studies ofphagocytosis by polymorphonuclear leukocytes: Use of emulsion to measure the initial rateof phagocytosis. .7. din. Invest. 51, 615-624.

UEDA, M. J., ITO, T., OHNISHI, S. & OKADA, T. (1981). Phagocytosis by macrophages. I.Kinetics of adhesion between particles and phagocytes. J. Cell Sci. 51, 173-188.

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(Received 12 January 1981)

phagocytosis by macrophages - journal of cell science

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