J. Cell Sci. 51, 189- aoi (1981) Printed in Great Britain Company of Biologists Limited 1981 PHAGOCYTOSIS BY MACROPHAGES II. THE DISSOCIATION OF THE ATTACHMENT AND INGESTION STEPS TADANAO ITO, MASAMICHI J. UEDA*. T. S. OKADA AND SHUN-ICHI OHNISHI Department of Biophysics, Faculty of Science, Kyoto University, Kyoto, Japan SUMMARY The phagocytic process of mouse peritoneal macrophages was dissociated, using bovine serum 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 the cell surface by treatment with a membrane-impermeable reducing agent, ascorbate. The validity of the assay method was tested under various conditions. The measurements piovided accurate and reproducible data. The phagocytic reaction was followed as a function of time and the rate constants for the attachment and ingestion steps were obtained from the initial phase. Both steps were highly dependent on temperature. Divalent cations in the incubation medium were essential for the attachment step but apparently had no effect on the ingestion step. The metabolic inhibitors, KCN and 2-deoxyglucose, inhibited both steps. Cytochalasin B inhibited both steps, while colchicine inhibited only the attachment step but apparently had no effect on the ingestion step. INTRODUCTION Phagocytosis is a fundamental cellular function that internalizes exogenous particles into the cytoplasm (Silverstein, Steinman & Cohn, 1977). After the attachment of particles to the cell surface, pseudopodia appear and extend to embrace the particulate object. The membrane pleats surrounding the particle ultimately fuse and encase the object within a phagocytic vesicle. The involvement of cytoplasmic contractile proteins in the extension of pseudopodia and the engulfment of particles has been suggested (Stossel & Hartwig, 1976). The phagocytic process can be divided experimentally into 2 discrete steps: attachment and ingestion of particles. The method used was direct microscopic counting of the number of particles attached to the cell surface and internalized into the cell (Allen & Cook, 1970; Michl, Ohlbaum & Silverstein, 1976; Rabinovitch, 1967). Since the number of cells counted is limited for technical reasons, the measurements are not accurate enough for a kinetic analysis of phagocytosis. A more accurate 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' particles was 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 the Michaelis-Menten type of mechanism has been carried out based on these data. However, this method cannot be used to discriminate between the attached and ingested particles. We have developed a new spectrophotometric method that can dissociate the phagocytic reaction into attachment and ingestion steps. The method employs electron spin resonance (e.s.r.) measurement of spin-labelled cholestanone dissolved into bovine serum albumin (BSA)-coated paraffin particles. The intensity of the signal of cell-associated particles gives a measure of the sum of the attached and ingested particles, and the intensity after treatment of the cells with ascorbate provides a measure of the ingested particles, since ascorbate reduces the spin label and destroys the e.s.r. signal, and because the cell membrane is practically impermeable to ascorbate at low temperatures. After experimental tests of the validity of the method, we have investigated the effect of temperature and various additives on 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 containing o-1 % (w/w) glucose buffered at ph 74 with 10 mm-.n-2-hydroxyethylpiperazine-2v v -2-ethanesulphonic acid (Hanks/HEPES/glucose). Preparation of particles for phagocytosis An emulsion of paraffin oil coated with BSA or opsonized with complement was prepared by 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 (fraction V, Reheis Chemical Co.). The 5 mm-0 tip of a 20 khz sonicator (Kaijo Denki Co.) was placed above the oil-water interface and emulsion was achieved at 250 ma of plate current for 10 min at o C. The density of the emulsion was about 088 g/cm' and all the cholestanone spin label remained in the emulsion. The spin label, a 7V-oxy-4',4'-dimethyloxazolidine derivative 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 cholestanone spin 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 particles were washed twice before opsonization. The washing was accomplished by centrifugation at 10000 g for 10 min, removal of the infranatant fluid and resuspension of the particles in Hanks/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 opsonized particles 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 (~ io 7 cells/ml) were preincubated in a 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 the incubation, 6 ml of iv-ethylmaleimide (1 min) in 145 mm-nacl was added to the cell suspension. For the assay of total associated particles, the cell suspension was centrifuged at 1500 g for 5 min at 4 C and the supernatant was discarded. The inside of the test tube was wiped with tissue paper to remove the particles remaining on the surface. The pellet was placed in a quartz capillary tube and the e.s.r. spectrum was measured at 22 C with a JEOLCO model ME-X spectrometer. The central peak height was read and used to quantitate the particles attached and ingested inside the cells. For the assay of ingested particles, the cell suspension was centrifuged at 400 g for 5 min at 4 C and the supernatant was discarded. The inside of the test tube was wiped as mentioned above. One ml of ascorbate solution was added to the pellet and it was kept for 90 min at o C. The ascorbate solution consisted of 07 ml of Hanks/HEPES/glucose containing 1-4 mmiy-ethylmaleimide and 0-3 ml of freshly prepared 300 mm-sodium ascorbate (ph adjusted to 7-4). The mixture was then centrifuged at 600 g for 5 min at 4 C. The pellet was taken into the 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 ascorbate and the almost complete impermeability of the cell membrane to ascorbate at low temperatures. Fig. 1, curve A shows the decay of the e.s.r. signal of cholestanone spin label contained in the BSA-coated paraffin particles when treated with ascorbate at o C. The intensity gradually decreased and became less than 5 % of the initial value after 90 min. On the other hand, when the particles were incubated with macrophages at 37 C and the cells were treated with ascorbate at o C, the signal intensity 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 signal in the time interval. The results therefore indicate that about 50% of the particles were located on the outside of the cells where ascorbate was accessible, and the rest of the particles were ingested inside the cells and protected from reduction by ascorbate. It is also shown that the spin label in the ingested particles was protected from various reducing agents in the cytoplasm. The above conclusion was confirmed by another experiment in which complementopsonized paraffin particles were used. After incubating macrophages with opsonized particles 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 incubated for the appropriate time at 37 C. Ascorbate was added to the cells and they were kept for 90 min at o C. The remaining e.s.r. signal was larger for the cells incubated for a longer time at 37 C. After incubation for 30 min, the e.s.r. signal intensity became 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 by ascorbate. Curve A, particles only; ascorbate was added at o C C to the particle suspension and the e.s.r. signal was measured at given times. Curve B, after incubation with macrophages; the cells were incubated with the particles at 37 C for 4 min and centrifuged. Ascorbate was added at o C to the cell pellet and the e.s.r. signal was measured at the times given in the text.» 0-6 - 10 20 30 Incubation time (min) Fig. 2. Increase in the number of ingested particles with incubation. Macrophages were incubated with the complement-opsonized particles at o C C for 60 min and centrifuged. The cells were resuspended in Hanks/HEPES/glucose, incubated at 37 C for various periods, and the e.s.r. spectrum was measured before treatment with ascorbate {A) or after (B). Ascorbate was added to the cells (at o C), which were kept for 90 min before the e.s.r. measurement. The signal intensity is plotted against incubation time at 37 C.
Phagocytosis by macrophages 193 particles were ingested into the cytoplasm and completely protected against ascorbate and endogenous reducing agents in the macrophages. The treatment with ascorbate did not affect cell viability. The cells treated with ascorbate for 90 min at o C were cultured overnight in a Falcon plastic dish containing Eagle's minimal essential medium supplemented with 6 % foetal calf serum under a humid atmosphere of 95 % air and 5 % CO 2 at 37 C. Almost all the macrophages were intact and adhered to the dish. These cells were also found to be viable by 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: irreversible attachment and ingestion. V v Particles in the medium > particles attached to the cell surface^) > particles ingested inside the cell (n 2 ). 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 that of the cells after treatment with ascorbate gives the number of ingested particles (n 2 ). The following equations hold, where t is time: dnjdt = V-v, (1) dn 2 /d* = v. (2) From equations (1) and (2) d(n 1 + n 2 )/d* = V (3) follows. When (n x + n^) was plotted against incubation time at 37 C, the curve initially followed a straight line and then gradually deviated (Fig. 3). Therefore, the slope of the linear region corresponds to the initial rate of irreversible attachment (J9. Within this time interval (4 min at 37 C), n x + n 2 = V,t (4) can hold. We represent the rate of ingestion, v, as v = kji lt (5) where k 2 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 the experimental values of n 2 are plotted together with the theoretical curve calculated using 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 8 Incubation time (min) Fig. 3. Time course of the increase in the number of cell-associated particles with incubation. Macrophages were incubated with the BSA-coated particles at 37 C for the indicated times and centrifuged. The e.s.r. signal intensity of the cell pellet is plotted against incubation time. 10 g 0-5 I I I 2 4 6 Incubation time (min) Fig. 4. Comparison of experimental number of ingested particles (O) with theoretical curve ( ), calculated using equation (5) with k t = 0-27 min- 1. The value at 4 min was fitted to the theoretical curve. The ordinate is the e.s.r. signal intensity after treatment with ascorbate of macrophages that had been incubated with the BSAcoated particles at 37 C for the times indicated.
Phagocytosis by macrophages 195 The fraction P is thus dependent only on k 2, whereas the total number of associated particles depends only on T^. Kinetic analysis of the initial rate, V l} 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 interest in the present study is to discriminate between irreversible attachment and ingestion and therefore such a kinetic analysis was not done. In routine assays, the phagocytic reaction was carried out for 4 min at 37 C, and the total number of associated particles and the fraction of ingested particles were 0-5 - - 100 0-3 50 0-1 10 20 30 40 Incubation temperature ( C) Fig. 5. Temperature dependence of the attachment and ingestion steps of phagocytosis. The cells were incubated with the BSA-coated particles for 4 min at the indicated temperatures and values of (f^ + Wi) ( ) and P(O) were obtained from the e.s.r. measurements, (n^ + n^) is given as a percentage of the value at 37 C C. I obtained from the e.s.r. signal intensity. Average values of {n^ + n^) and P were about 0-5 mg paraffin emulsion per io 7 cells and 0-4, respectively. The absolute values varied with different batches of cells prepared separately. This appears to be due mainly to differences of cell viability. A series of experiments was carried out on cells derived from the same batch. The reproducibility of data was quite good to within 95%. The effects of drugs, divalent cations and temperature were expressed as the ratio to the control («i + «2 ) was calculated to measure the attachment step and 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 ingestion steps of the phagocytic reaction. Both (n x + «8 ) and P decreased markedly with decreases in temperature. For example, the ratio of (n l + n 2 ) at 25 C to that at
196 T. Ito, M. J. Veda, T. S. Okada and S. Ohnishi 37 C was 0-19 and the ratio of P at 25 C to that 37 C was 0-34. The activation energy of the ingestion rate, k it was determined to be 22 kcal moh 1 from equation (7). Effect of metabolic inhibitors Since phagocytosis is known to be an energy-dependent process, the effects of KCN, an inhibitor of oxidative metabolism and 2-deoxyglucose, a glycolytic inhibitor, were investigated. The results showed that these metabolic inhibitors inhibited both 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 n,+ n 2 P A KCN () B 2-Deoxyglucose 100-50- 100-0-3 0-2 - 0-2 I I 001 0-5 10 50 10 50 Concentration (nrim) Concentration Fig. 6. Effect of metabolic inhibitors on the attachment and ingestion steps of phagocytosis. Macrophages were preincubated at 37 C C 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. (n l + n i ) to 24% and P to 63% of the control. Glucose added to 5 mm did not affect the phagocytic reaction. These results indicates that the rates of irreversible attachment and ingestion were decreased to 48 and 46% by 1 mm-kcn and to 24 and 47 % by 5 mm-2-deoxyglucose, respectively. Effect of divalent cations In order to investigate the effect of divalent cations, the cells were preincubated at 37 C in Hanks/HEPES/glucose containing only the indicated cation, and then incubated with BSA-coated paraffin particles. The results are summarized in Table 1, which shows, firstly, that the divalent cations are indispensable for the attachment step. Omission of divalent cations at various concentrations markedly reduced the
Phagocytosis by macrophages 197 Table i. Effect of divalent cations on attachment and ingestion steps of phagocytosis Divalent cation Mg 1+ Ca«+ Mn'+ Co«+ Concentration (mm) i-o O-2 i-o 0-05 o-oz 0-2 (Vr + n,) (». + ",), 063 0-36 o-34 071 0-62 0-69 0-59 p Po I-I I-I I-I I-I I-O I-I 0-05 0-4 0-70 i-o O-I5 0-44 I-I Hanks/HEPES/glucose normally contains i-2mm-mg* + and o-8 mm-ca* +. The medium was depleted of these divalent cations and the appropriate cation was added to the medium as required. Macrophages were preincubated in the medium and mixed with the spin-labelled paraffin particles that had been emulsified in divalent-free medium. The BSA coated on the particles was dialysed on divalent-free medium before use. Incubation was for 4 min at 37 C (nj +«,) and P values are given as the ratios to the control (i.e. in 1-2 mm-mg' + and o-8 mm-ca l+ ). The subscript c means control. 0-8 0-6 0-4 5 10 Incubation time (mm) Fig. 7. Effect of divalent cations on the increase in the number of ingested particles with incubation. Macrophages were incubated with the BSA-coated particles at 37 C for 4 min and centrifuged. The cells were resuspended in Hanks/HEPES/ glucose containing 1-2 mm-mg t+ and o-8 mm-ca 1+ ( ), or 0-5 mm-edta without divalent cations (O). and incubated at 37 C for various periods of time. Ascorbate was 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.
198 T. Ito, M. J. Veda, T. S. Okada and S. Ohnishi - 100 0-4- - + 10 Concentration - 100 + c 0-2 - 10 10 Concentration (J/M) Fig. 8. Effect of cytochalasin B (A) and colchicine (B) on the attachment and ingestion steps of phagocytosis. Macrophages were preincubated in Hanks/HEPES/glucose containing the indicated concentrations of drugs at 37 C for 10 min and then incubated 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 ingestion step. P/P c remained at i-o to 11. This conclusion was ascertained by more-direct experiments. After incubation with the BSA-coated particles at 37 C for 4 min, the cells were collected by centrifugation and resuspended in Hanks/HEPES/glucose containing 05 mm-edta but no divalent cations. In a control experiment, the cells were resuspended in normal Hanks/HEPES/glucose that contained 12 mm-mg 2+ and o-8 mm-ca 2+. The cell suspension was incubated at 37 C for the appropriate time, centrifuged, and treated with ascorbate. The remaining e.s.r. signal increased with incubation time. It was remarkable that the time course for the cells incubated in the absence of divalent
Phagocytosis by macrophages 199 cations could be superimposed on that of the control as shown in Fig. 7. This result strongly 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 steps of 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 ingestion step (Fig. 8). Colchicine inhibited the attachment step but relatively weakly. DISCUSSION Possible steps in the whole process of phagocytosis are presented in Fig. 9. These include reversible (a) and irreversible (b) attachment of particles, extension of pseudopodia (c), and membrane fusion and ingestion (d). The stage at which ascorbate cannot attack the particles is E. In the present analysis, the overall step from A to C was treated as attachment and the sequential steps from C to E as <4 E Fig. 9. Possible steps in phagocytosis, (a) Reversible attachment; (6) irreversible attachment of a particle, (c) Membrane envelopment around the particle, (d) Membrane fusion and ingestion of the particles into the cytoplasm. Stages A to E are described in the text. D ingestion. The overall step from A to C could be analysed including an intermediate state, B, as done by Weisman & Korn (1967), Stossel (1973) and also by the present authors (Ueda, Ito, Ohnishi & Okada, 1981). However, our main interest in the present analysis 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 envelopment and fusion. The results suggest that some rearrangements of cell-surface receptors are involved in the irreversible attachment step, since lowering the temperature, addition of metabolic inhibitors, and cytochalasin B inhibited the step. The ingestion step consists 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 important role in the extension of pseudopodia (Stossel & Hartwig, 1976). The inhibition of the ingestion step by cytochalasin B and metabolic inhibitors is therefore quite understandable because they impair the function of cytoplasmic contractile proteins. The role of divalent cations is unique since they were indispensable for irreversible attachment but had no effect on the ingestion step. When the cell-particle complex was 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 that extension of pseudopodia and membrane fusion will occur without external divalent cations. Stimulation of the rate of particle uptake by divalent cations has also been indicated from Michaelis-Menten type analysis of the phagocytic reaction of granulocytes and rabbit alveolar macrophages (Stossel, 1973). The present analysis gives further insight into the role of divalent cations. Rabinovitch (1967) has dissociated the steps of attachment and ingestion in mouse peritoneal macrophages by experiments using optical microscopy. Both steps were dependent on temperature, in qualitative agreement with our results. However, the requirement of divalent cations in the 2 individual steps was the opposite to the findings of the present results. Michell et al. (1969) have observed that 2-deoxyglucose had no inhibitory effect on the capacity of mouse peritoneal macrophages to phagocytize latex or zymosan particles. The discrepancy with our results may be due to different experimental conditions. These authors observed phagocytosis when the uptake of particles proceeded virtually to completion, while we measured the initial rate. The spin-label assay can provide accurate data on the 2 individual steps in phagocytosis. 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 values from ~ io 7 cells, in comparison with ~ io 2 cells in the microscopic method. Detailed information on individual steps would facilitate our understanding of the whole phagocytic reaction in molecular terms. REFERENCES ALLEN, J. M. & COOK, G. M. W. (1970). A study of the attachment phase of phagocytosis by murine macrophages. Expl Cell Res. 59, 105-116. BOYES, E. A., OLD, L. J. & CHAROULIKOV, J. (1964). Cytotoxicity test for demonstration of antibodies. Meth. med. Res. 10, 39 47. ICHIKAWA, Y., PLUZIK, D. H. & SACHS, L. (1967). Feedback inhibition of the developement of 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). Measurement of 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 inhibits Fc 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 phagocytosis by macrophages. Expl Cell Res. 46, 19-28.
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