Journal of Cell Science 101, 907-913 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

907

The macrophage capacity for phagocytosis

GREGORY J. CANNON and JOEL A. SWANSON*

Department of Anatomy and Cellular Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA

•Author for correspondence

Summary

Murine bone marrow-derived macrophages, whichmeasure 13.8 ± 2.3 /an diameter in suspension, caningest IgG-opsonized latex beads greater than 20 /ondiameter. A precise assay has allowed the determinationof the phagocytic capacity, and of physiological para-meters that limit that capacity. Ingestion of beads largerthan 15 /mi diameter required IgG-opsonization, andtook 30 minutes to reach completion. Despite thedependence on Fc-receptors for phagocytosis of largerbeads, cells reached then* limit before all cell surface Fc-receptors were occupied. The maximal membranesurface area after frustrated phagocytosis of opsonizedcoverslips was similar to the membrane surface arearequired to engulf particles at the limiting diameter,indicating that the capacity was independent of particleshape. Vacuolation of the lysosomal compartment with

sucrose, which expanded endocytic compartments,lowered the phagocytic capacity. This decrease wasreversed when sucrose vacuoles were collapsed byincubation of cells with invertase. These experimentsindicate that the phagocytic capacity is limited by theamount of available membrane, rather than by theavailability of Fc-receptors. The capacity was alsoreduced by depolymerization of cytoplasmic micro-tubules with nocodazole. Nocodazole did not affect thearea of maximal cell spreading during frustratedphagocytosis, but did alter the shape of the spread cells.Thus, microtubules may coordinate cytoplasm forengulfment of the largest particles.

Key words: macrophage, phagocytosis, Fc-receptors,microtubules.

Introduction

Macrophages can ingest great quantities of particles,yet not so much that they burst. When plated onto flatsurfaces opsonized with IgG, they engage that surfaceas if to engulf it, a process termed frustrated phagocyto-sis (Henson, 1971; Takemura et al., 1986), and spreadto some limit. It may be that the macrophage capacityfor phagocytosis or its extent of spreading duringfrustrated phagocytosis is limited by the number ofavailable phagocytic receptors, and when all of themhave been internalized or engaged phagocytosis stops.Alternatively, some cellular structure which changesduring the process may approach a limit, and that limitdefines satiety.

The goals of the present experiments were todetermine the phagocytic capacity of murine bonemarrow-derived macrophages, to compare that capacityto frustrated phagocytosis, and to identify factors thatlimit those capacities. Capacity was measured usingpolystyrene beads that range in size from those thatcould easily be ingested by one cell to those that weresignificantly larger than the cell itself. The cell and beadconcentrations were adjusted so that the usual con-dition consisted of one cell attempting to phagocytoseone bead. Determining the largest bead that the

macrophage could engulf under various conditionsallowed for the examination of several interestingcharacteristics of phagocytosis. Cell surface area at thephagocytic limit was similar to that reached afterfrustrated phagocytosis. Fc-mediated phagocytosisstopped before all Fc-receptors were engaged orinternalized, indicating that cell surface membranebecame limiting. Moreover, the phagocytic capacitywas lowered when microtubules were depolymerized.Comparisons with frustrated phagocytosis suggestedthat the radial symmetry of the phagocytic responseenhanced the phagocytic capacity.

Materials and methods

CellsMurine bone marrow-derived macrophages were obtained bythe method of Swanson (1989b). Bone marrow was removedfrom femurs of female ICR mice (Charles River, Cambridge,MA) and was washed 3x in cold Dulbecco's modifiedessential medium plus 10% heat-inactivated fetal bovineserum (DME-10F). Cells were resuspended and plated at 2 X105 cells/ml on 100 mm Lab-Tek dishes in 25 ml or on 60 mmLab-Tek dishes in 10 ml of complete bone marrow medium(DME plus 30% L-cell-conditioned medium plus 20% heat-inactivated fetal bovine serum). The cells were incubated at

908 G. J. Cannon and J. A. Swanson

37°C under 5% CO2 for 6 days, and the macrophages adheredto the bottom of the dish during this period. Macrophageswere harvested from dishes after brief washing with cold,sterile, divalent cation-free, phosphate-buffered saline (PD:137 mM NaCl, 3 mM KC1, 7 mM phosphate buffer, pH 7.4).The resuspended cells were plated onto 12 mm coverslips in24-well Costar dishes at 1 x 105 cells/ml. After 30 minutes at37°C, the PD was replaced with DME-10F. Cells were usedwithin the next two days. Greater than 95% of the cells weremacrophages, as determined by their ability to phagocytoseopsonized sheep red cells.

Macrophages resuspended from Lab-Tek dishes into coldPD were examined microscopically (magnification, X500).Diameters of 83 rounded, unspread cells were measured usinga calibrated eyepiece graticle.

Beads2 x 106 polystyrene polybeads of diameter 21.1 fjm (Poly-sciences Inc., Warrington, PA) were incubated in 10 mg/mlbovine serum albumin (BSA, Sigma Chem. Co., St. Louis) inPD for 1 hour at 4CC. The beads were then washed 5 times inPD, then resuspended in 1 ml PD. Rabbit anti-bovinealbumin, IgG fraction (anti-BSA IgG, Cappel, West Chester,PA), was added to a final dilution of 1:500, then incubated for30 minutes at 37°C and 10 minutes at 4°C. The beads werethen washed 3 x in 1 ml PD and recounted. The 21.1 jumbeads had a considerable variance in size (4.09 /an, accordingto the manufacturer) with a range of 13 jjm to >30 fim indiameter, and this made the phagocytosis assay possible.

For other phagocytosis assays, 21.1 /m\ BSA-coated beadswere incubated in 6 mg/ml 2,4-dinitrobenzene sulfonate(DNBS, Aldrich Chem. Co., Milwaukee), in 4% K2CO3 for 1hour, then washed 3 x in 1 ml PD. The beads were thenincubated in anti-DNP IgG before or after they came incontact with the macrophages.

Basic phagocytosis assayA quantitative measurement for the phagocytic capacity ofmacrophages was obtained by allowing the cells to engulflarge latex beads, then using immunofluorescence to labelbeads that were not internalized, and measuring how manybeads of a given size interval were positively labeled.

Fresh DME-10F (1 ml) was added to day 7 or day 8macrophages. 2 x Mr beads (21.1 fan diameter) were addedto each well. Cells were allowed to phagocytose the beads forone hour. Dishes were then placed on ice, washed gently 3 xin cold PD, the wells aspirated dry, and rhodamine-labeledgoat anti-rabbit IgG (rhodamine-GAR: heavy and light chainspecific, Cappel), 300 /A at 1:50 in PD, was added. The cellswere then incubated on ice for 30 minutes and washed 3 x inPD. Control experiments showed that only beads which hadboth BSA and anti-BSA on them could be labeled with therhodamine-labeled secondary antibody (data not shown).Even though 30 minutes at 4°C was allowed for therhodamine-labeling step of the assay, beads were completelylabeled in less than 10 minutes at that temperature.

For microscopic analysis, the coverslips were inverted,mounted on a glass slide on top of glass coverslip fragments,and the cavity filled with PD and sealed with valap to preventdrying (Swanson, 1989a). The cells were studied with a ZeissPhotomicroscope in equipped for epi-illumination of fluor-escent specimens. To measure bead diameters, a Wert xlOocular micrometer was calibrated with a stage micrometer.The divisions on the ocular micrometer were 1.8 micrometersapart at x500. For each bead, the diameter was measured andthe bead scored as plus or minus for rhodamine fluorescencewith rhodamine filters. Only in cases where there was one

bead per macrophage, and one macrophage per bead, weremeasurements taken. Although a phase 3 lens was used toview the cells, a condenser lens without phase rings was usedduring bead diameter measurement to avoid optical distortionof bead dimensions. The diameter of the beads could bemeasured to the nearest micrometer with considerableaccuracy. For each 1.8 /.an gradation, 10 beads were scored forfluorescence.

Determination of the parameters of phagocytosisAll of the experiments described in this section were executedin their entirety at least twice.

Time course of phagocytosisThe beads were assayed as above, except that phagocytosiswas allowed to proceed for shorter periods of time: 5,10, 20,30 and 60 minutes. Coverslips were removed from the dishand placed in a new dish on ice with 1 ml PD in each well.Rhodamine-GAR was added after the cells were chilled andwashed.

Phagocytosis of opsonized beadsBSA/anti-BSA-coated beads, BSA/DNP-coated beads (noanti-DNP), and BSA/DNP beads that had been opsonizedwith rabbit anti-DNP IgG were added to two wells. After onehour of phagocytosis, anti-DNP was added to the unopso-nized bead wells (300 /A of 50 j<g/ml in PD) for 30 minutes onice, while the others were kept in PD. After 3 washes, all wereincubated in the rhodamine-GAR.

Effect of nocodazole on phagocytic capacityMacrophages were incubated in 10 JJM nocodazole (SigmaChemical, St. Louis, MO ) in DME-10F for one hour before21.1 /un beads were added. The phagocytosis and labelingprocedures remained the same.

Effect of cell vacuolationMacrophages were incubated overnight (15 hours) in 10mg/ml or 20 mg/ml sucrose in DME-10F according to Swansonet al. (1986). Phagocytosis and labeling protocols wereunchanged. For invertase recovery, cells that had beenincubating in 20 mg/ml sucrose for 15 hours were washed twicein medium, then 1 ml of 0.5 mg/ml invertase (Sigma Chem.Co.) in medium was added for 2, 4 or 6 hours before thephagocytosis assay was performed. In control experiments,vacuolated cells were maintained in sucrose for 4 hours beforeallowing phagocytosis.

Macrophage spreading on opsonized coverslipsThe extent of cellular spreading of macrophages that engagein frustrated phagocytosis was determined. 12 mm coverslipswere treated with DNP according to the method of Michl etal. (1979). Coverslips were opsonized with 50 /ig/ml anti-DNPIgG in PD for 30 minutes and then washed 3 x in PD.Macrophages that had been chilled and resuspended fromLab-Tek dishes were plated onto the coverslips at 1-2 x104/well. After 1 hour of spreading, the cells were chilled onice and then incubated with 300 [A rhodamine-GAR (1:100)for 30 minutes to label areas of the coverslip which were notmasked by the macrophages. The cell diameters weremeasured with the ocular micrometer.

For other studies, coverslips coated with BSA wereopsonized with anti-BSA IgG for 30 minutes, then washedthoroughly before plating macrophages. Macrophages resus-pended from Lab-Tek dishes were plated onto coverslips inRinger's saline ± 10 /JM nocodazole.

The macrophage capacity for phagocytosis 909

Erythrocyte binding to macrophagesSheep red blood cells (SRBC: Cappel) were opsonized withgoat anti-SRBC IgG (Cappel) at a final dilution of 1:1000 (30minutes 37°C, 10 minutes 4°C, washed 3 X in PD).Macrophages that had phagocytosed test beads were incu-bated for 1 hour with 3 x 106 opsonized SRBC. Cells weresubsequently chilled and incubated in rhodamine-GAR. Inseparate experiments, opsonized SRBC were added tomacrophages 1 hour after plating onto opsonized coverslips(DNP-anti DNP). SRBC were incubated with the macro-phages for 15 minutes at 4°C, then unbound SRBC werewashed away. The number of red cells on the surface of 100randomly selected macrophages was measured.

Measurements of macrophage area and shapeMacrophages were allowed to spread for 40 minutes ontocoverslips opsonized with anti-BSA IgG ± 10 /tM nocodazole.Cells were fixed with 3.7% formaldehyde plus 0.25 M sucrose,0.5 mM EDTA, 1 mM EGTA, 20 mM HEPES, pH 7.4, thenwere washed and observed by phase-contrast microscopy.Video images of 35 cells for each condition were recorded anddigitized for image processing. Outlines of cells were tracedusing interactive software; these tracings were used togenerate a binary mask for each cell, then these binaries wereanalyzed to measure area (A), perimeter (P) and the shapeparameter (P2/4;rA) for each cell using the image processingcapabilities of a Tracor Northern TN 8500 system (NoranInst., Middletown, WI).

Results

Phagocytic capacityThe phagocytosis assay measured the percentage oflatex beads within a given size range that could beengulfed by macrophages. Only instances in which onemacrophage engaged one bead were acceptable formeasurements. Internalized beads were identified assuch by their inaccessibility to fluorescently labeledantibodies against their surfaces. Beads bound to cellswere almost always completely dark or brightly fluor-escent. Exceptions occurred when beads were partiallyengulfed and thus only partially labeled; such beadswere scored positive for fluorescence (i.e. they hadfailed to engulf the bead). The basic assay to determinethe phagocytic capacity of bone marrow-derived macro-phages was performed as a separate experiment and asa control for each of the subsequent experimentalvariations. The data were averaged and expressed asthe percentage of positively labeled beads in each beadsize category (Fig. 1). An operative definition for thephagocytic limit was given as the bead diameter atwhich 50 per cent of the beads scored positive forrhodamine labeling. In order to determine this point, alogit transformation was performed on the data to makethe sigmoidal curve linear, and then first-order re-gression analysis was performed. In the control con-dition, the 50% mark occurred at 19.8 /im, with a 95%confidence interval of 17.0 jim to 22.5 ̂ urn. Macrophagesresuspended from a culture dish assume a sphericalmorphology. The diameters of resuspended macro-phages were found to be 13.8 ± 2.3 /xm (s.d.). Thecalculated phagocytic capacity of 19.8 fim indicates that

r4 18 20Bead diameter (/mi)

22 24

Fig. 1. Determination of the macrophage capacity forphagocytosis. A mixture of opsonized particles of varyingdiameter was added to macrophages. After 60 min forphagocytosis, the cells were chilled and incubated withrhodamine-labelled goat anti-rabbit IgG, to decorateexposed particle surfaces. Ten beads of each size class, thatalso contained an associated macrophage, were scored aspositive (external) or negative (internalized) for rhodaminefluorescence. Data from several experiments were pooledto obtain the values shown. The capacity was operationallydefined as the bead diameter at which half the beads wereengulfed. The schematic drawings indicate the principle ofthe labelling procedure, with bristles indicating the exposedsurface of the larger particle (left).

14 16 18 20Bead diameter (/mi)

24

Fig. 2. Time-course of the phagocytic response.Macrophages were provided opsonized particles for 5 ( • ) ,10 (A), 20 (A), 30 (•) or 60 (•) min at 37°C beforechilling and labelling with fluorescent secondary antibodies.Cells reach their final capacity with 30 minutes ofphagocytosis.

those cells are capable of ingesting particles 1.44 timestheir diameter, or 3 times their volume.

The time course of phagocytosis was determined byvarying the time that the macrophages were exposed tothe beads at 37°C. As Fig. 2 indicates, macrophagesreached their phagocytic capacity by the 30 minute time

910 G. J. Cannon and J. A. Swanson

point (50% =20.8, P>0.5). The phagocytic size limit at20 minutes was already not significantly different fromthe control values (50%=18.4 jm\, P<0.40).

To examine the role of opsonization in the phago-cytosis of larger beads, BSA-coated beads were labeledwith dinitrobenzene sulfonate (DNBS) before incu-bation with the cells, and phagocytosis was measured bychilling and labelling the beads with anti-DNP and thenrhodamine-GAR. Macrophages phagocytosed DNP/anti-DNP coated beads (50% = 19.1 fzm, P>0.50) to thesame extent as they did BSA/anti-BSA coated beads,but unopsonized beads had a 50% point of 14.0 ftm(/><0.001, data not shown). This implicates receptor-ligand interactions as necessary for the phagocytosis oflarger beads.

Measurements of cellular spreadingWe compared the phagocytic capacity for opsonizedbeads with the extent of macrophage spreading onopsonized substrata. This frustrated phagocytosis(Henson, 1971; Michl et al., 1979; Takemura et al.,1986) is analogous to the phagocytosis of an infinitelylarge particle. A significant difference in values ob-tained for phagocytosis of beads versus those forspreading would indicate that the cells can distinguishshapes of particles during phagocytosis. Due to thetightness of the seal that the spread cells formed withthe coverslip, and the fact that cells that were nottouching any other cells would spread into circularprofiles (Fig. 3A), a simple measurement of thediameter of the circular profile permitted estimation ofcell spreading and surface area. Since cells eithertended to clump together in suspension, or to contacteach other as they spread, only the 10 largest, isolatedand circular macrophage profiles on each coverslip weremeasured. The average diameter of the 10 largest cellson coverslips from several different cultures of macro-phages was 44.3 [im, with a standard deviation of 3.8,um. A circle with this diameter has an area of 1,541,um2. To account for both the upper and lower faces ofthe cell, the area should be doubled to yield a cellsurface area of 3,080 fim2. This estimate is a minimumvalue for the surface area, since it considers the cell asan infinitely thin disk. It is a fair estimate, however,since cell ruffling is considerably reduced in thiscondition, and the cells are very flat (Fig. 3A).Moreover, our estimate is in good agreement with otherdeterminations of surface area obtained in thioglyco-late-elicited peritoneal macrophages (Phaire-Wash-ington, 1980). This limit is similar to the surface arearequired to ingest the largest beads. To engulf a bead of19.8 ;Um, the macrophage plasma membrane must reacha surface area of at least 2,463 ̂ m2 (2 x 1232 ̂ im2). Thissimilarity between bead phagocytosis and frustratedphagocytosis indicates that at their limit these twoprocesses attain the same total cell surface area, andthat macrophages do not discriminate particle shapes inthese processes. The difference of 600 (im2 may beexplained by the difference in the way the limits aredefined. For frustrated phagocytosis we measured the10 largest circular cell profiles, essentially identifying

Fig. 3. Nocodazole treatment does not inhibit frustratedphagocytosis, but alters the symmetry of macrophagespreading. Macrophages were plated onto opsonizedcoverslips for 40 min with (B) or without (A) 10 f.annocodazole, then were fixed for microscopy. Bar, 20 fan.

champions, whereas for bead phagocytosis we attainedthe half maximal bead size for phagocytosis, whichmeasures the capacities of the whole population. Thelatter method would be expected to yield lowernumbers.

Fc receptors on the surfaces of the macrophageAlthough it appears that membrane availability is thelimiting factor with respect to phagocytic capacity,another reason why the cells might not be able to ingestmore could be that they deplete from their surfaces allreceptors for binding with the opsonized particles. Totest this, opsonized sheep erythrocytes were added toeither normally spread macrophages that had phago-cytosed a large bead or macrophages that wereengaging a coverslip in frustrated phagocytosis. Asshown in Fig. 4, macrophages spread onto opsonizedsurfaces could bind red cells on their upper surface. A

Fig. 4. Frustrated phagocytosis does not deplete the cellsurface of Fc-receptors. Macrophages were allowed toengage the opsonized coverslip for 60 min, then werechilled and provided opsonized erythrocytes. Macrophagescontinued to bind erythrocytes even after spreading fully.

count of erythrocytes per 100 cells yielded 15 (±6)bound erythrocytes per macrophage. Similarly, macro-phages engaging large, opsonized beads could also bindopsonized erythrocytes (data not shown). These resultsindicate that the phagocytic capacity is not limited bythe availability of receptors.

Role of internal compartmentsThe cell surface areas reached by phagocytosis wereconsiderably greater than the reported macrophagemembrane surface area (825 ^ m ; Steinman et al.,1976), indicating that internal membranes are recruitedto the plasma membrane during phagocytosis. Weasked how expanding internal membranous compart-ments affected the engulfment of larger beads. To dothis, the endocytic and lysosomal compartments werevacuolated during a 15 hour incubation in sucrose.Macrophages lack invertase, so pinocytosed sucroseaccumulates in lysosomes, where it expands thatcompartment osmotically (Cohn and Ehrenreich,1969). Cells were incubated overnight in variousconcentrations of sucrose and then the phagocytosisassay was performed (Table 1). The ability of macro-phages to ingest beads was drastically reduced after

Table 1. Phagocytic indices following vacuolation withsucrose or invertase-mediated sucrose vacuole collapse

The macrophage capacity for phagocytosis 911

overnight vacuolation in 30 mg/ml and 20 mg/mlsucrose. 10 mg/ml sucrose also had an appreciableeffect, reducing the 50% labeled value to 16.4 urn(P<0.02).

To determine if this decrease in the size limit was notdue to general impairment of cells, the vacuolation wasreversed with invertase. Invertase degrades sucrose toits component monosaccharides, which exit lysosomesand permit shrinkage of the lysosomal compartment(Cohn and Ehrenreich, 1969; Knapp and Swanson,1990; Swanson et al., 1986). Cells were vacuolated insucrose for 15 hours and then washed in medium. Theywere then incubated in 0.5 mg/ml invertase for up to 6hours (Table 1). Within 2 hours the phagocytic capacityhad nearly returned to pre-vacuolation levels. A smallincrease was seen at the 4 hour and 6 hour time points.The invertase-treated cells approached but did notreach pre-vacuolation levels.

Phagocytosis without microtubulesTo determine if microtubules were necessary forphagocytosis of the beads, cells were incubated in 10L*M nocodazole, which causes reversible depolymeriz-ation of macrophage microtubules (Swanson et al.,1987), for 1 hour before and during incubation with thebeads. Nocodazole produced a small but significantdecrease in the amount the cells could ingest (50% =16.9 Mm, P<0.05).

Because depolymerization of cytoplasmic micro-tubules reduced the phagocytic capacity, we comparedthis response to frustrated phagocytosis and found thatnocodazole did not limit the extent of spreading, butinstead made that spreading response irregular.Whereas the usual frustrated phagocytic responsecreated circular spreading profiles, with the nucleuscentrally placed, macrophages spreading in nocodazoleformed more irregular shapes (Fig. 3). We quantifiedthese observations by tracing the profiles of cells spreadonto opsonized coverslips for forty minutes in thepresence or absence of 10 /*M nocodazole. Using digitalimage processing, we determined spreading area andthe deviation of the profile from a circle (Table 2). Cellswere not significantly different in spread areas but weresignificantly different in shape. This indicates thatphagocytosis of the largest particles is enhanced by amicrotubule-dependent organization of cytoplasm.

Table 2. Macrophage area and shape after frustratedphagocytosis

Condition

Sucrose (mg/ml)in overnightincubation

102030202020

of cells

Invertasetreatment

(h)

246

50%

[an

16.414.9

<14.017.618.418.7

Phagocytosis

P

(<0.02)(<0.001)(<0.001)(<0.10)(<0.20)(<0.40)

Cells

ControlNocodazole

Profile

Area Shape

844<5±552 1.28±0.309382±952 2.25±0.26

Area and shape are determined from binary images, obtained byinteractive image processing. Areas are reported as number ofpixels per cell. Shape is expressed as perimeter (P) squared,divided by 4JI times the area (A). (P2/4^A). A circle would have ashape index of 1.0.

912 G. J. Cannon and J. A. Swanson

Discussion

Measurements using the phagocytic assay describedhere indicate that frustrated phagocytosis and particlephagocytosis are limited in similar ways, but aredifferent from cell spreading onto unopsonized sur-faces. Grinnell and Geiger (1986) described the phago-cytosis by fibroblasts of various sizes of fibronectin-coated beads and found that fibroblasts could phagocy-tose 6 /xm diameter beads easily, but could not engulfbeads larger than 12-14 /an diameter. In the light ofearlier work in which prior spreading on fibronectin-coated surfaces inhibited phagocytosis of fibronectin-coated beads (Grinnell, 1980), they concluded thatphagocytosis and cell spreading on flat surfaces werefundamentally the same process, which was limitedeither by the number of fibronectin receptors or bysome other physical constraint.

We found that particle capacity was increased byopsonization, but was not limited by Fc-receptoravailability. Macrophages at their phagocytic capacity,either with a bead inside or spread to their limit,remained capable of binding opsonized erythrocytes ontheir upper surfaces. The limit instead appeared to be inthe amount of cell surface membrane available forspreading. Macrophages in suspension, or plated ontounopsonized surfaces, contain prominent ruffles ofmembrane. When engaging an opsonized surface,ruffling membrane moves along that surface andflattens against it. As the limit is approached, surplusmembrane is recruited into the phagocytic response,and the cell surface is drawn smooth (Fig. 5). The limitseems therefore set by the amount of availablemembrane. An alternative explanation has beenoffered by Rabinovitch et al. (1975), that frustratedphagocytosis is not limited by spreading, but rather by aparalysis of Fc-receptors for phagocytosis. In theirstudies, murine peritoneal macrophages plated ontoopsonized coverslips could bind but not ingest opso-nized erythrocytes. They argued that this inhibition wasnot a consequence of cell spreading because otherconditions which increased cell spreading did not inhibitphagocytosis. It therefore remains possible that macro-phage frustrated phagocytosis is limited by a specificinactivation of Fc-receptor function, an inhibitionmediated by receptor ligation.

Our estimates of the cell surface area at capacity aresimilar for both bead phagocytosis and frustratedphagocytosis, and are also similar to values reported inearlier studies of phorbol ester-stimulated spreading(Phaire-Washington et al., 1980). Those surface areasare greater than those measured in unstimulatedmacrophages (825 /zm2; Steinman et al., 1976), indi-cating that intracellular membranes are brought to thesurface during phagocytosis. Occupation of internalmembranes, such as occurred during sucrose vacuo-lation, decreased the phagocytic capacity reversibly,indicating that these membranes contribute to thespreading. It remains possible, however, that macro-phage surface area does not change during phagocyto-sis; that the highly folded surface simply smooths out to

Fig. 5. The extent of cytoplasmic reorganization duringphagocytosis of the largest particles or during frustratedphagocytosis. (A) The macrophage in suspension has amean diameter of 14 fan, and an extensively ruffledsurface. Macrophages at their limits of particlephagocytosis (B) or frustrated phagocytosis (C) are muchlarger than unfed cells, and contain very few surfaceruffles. The phagocytic capacity may therefore be limitedby the amount of available cell surface membrane.

enclose particles, and that filling endocytic compart-ments with sucrose or smaller particles depletes the cellsurface of membrane which would otherwise beavailable for the spreading response.

The similar limits of particle phagocytosis andfrustrated phagocytosis indicate that they are notaffected by particle shape or by the size of thephagosomal lamellipod. The advancing edge of thepseudopod decreases beyond the equator of a sphericalparticle, but continually increases on a flat surface.Pseudopod advance therefore appears to be regulatedlocally, by the segmental interactions between Fc-receptors and the opsonized surface, and with the limitof advance set by the amount of available surfacemembrane.

That both particle phagocytosis and frustrated phago-cytosis proceed in the absence of cytoplasmic micro-tubules suggests that the process is independent of thiscytoskeletal element. Macrophages plated onto op-sonized surfaces in the presence of nocodazole engagethose surfaces in a phagocytic response, and reach thesame spreading surface area as control cells. However,the phagocytic capacity for particles is diminished bynocodazole treatment, indicating that microtubulescontribute to particle phagocytosis in some way. Wesuggest that microtubules provide a coordinating func-

The macrophage capacity for phagocytosis 913

tion. Such coordination is indicated by the strikingsymmetry of cell spreading during frustrated phago-cytosis. Macrophages spread into nearly perfect circles,with the nucleus centrally placed and with microtubulesradiating to the cell periphery from a central point. Inthe absence of microtubules, such spreading may beinitially circular, but soon rearranges to an irregularprofile. The radial symmetry that is lost duringnocodazole treatment may reflect a process thatcoordinates pseudopod closure around large particles.Without microtubules, uncoordinated pseudopod ad-vance around particles would produce irregularlyshaped gaps that close less readily than the small,circular gaps left by symmetrical spreading.

In contrast to Fc-mediated phagocytosis (Bhisey andFreed, 1971), complement-mediated phagocytosis inmacrophages is sensitive to microtubule-destabilizingdrugs (Wright and Silverstein, 1982). Complement-mediated phagocytosis therefore may be more similarto simple spreading processes, which are sensitive tonocodazole, than to Fc-mediated phagocytosis.Measurements of the phagocytic capacity of comp-lement-opsonized particles might be instructive in thisregard.

The authors gratefully acknowledge the assistance andadvice of Philip Knapp and Esther Racoosin. This work wassupported by the NIH (CA44328).

References

Bhisey, A. N. and Freed, J. J. (1971). Altered movement ofendosomes in colchicine-treated cultured macrophages. Exp. CellRes. 64, 430-438.

Cohn, Z. A. and Ehrenreich, B. A. (1969). The uptake, storage andintracellular hydrolysis of carbohydrates by macrophages. J. Exp.Med. 129, 201-225.

Grinned, F. (1980). Fibroblast receptor for cell adhesion: studies onthe interaction of baby hamster kidney cells with latex beads coated

by cold insoluble globulin (plasma fibronectin). / . Cell Biol. 86,104.

Grinnell, F. and Geiger, B. (1986). Interaction of fibronectin-coatedbeads with attached and spread fibroblasts. Exp. Cell Res. 162,449.

Henson, P. M. (1971). Interaction of cells with immune complexes:adherence, release of constituents, and tissue injury. J. Exp. Med.134, 114s-135s.

Knapp, P. E. and Swanson, J. A. (1990). Plasticity of the tubularlysosomal compartment in macrophages. J. Cell Sci. 95, 433-439.

Mlchl, J., Pieczonka, M. M., Unkeless, J. C. and Silverstein, S. C.(1979). Effects of immobilized immune complexes on Fc- andcomplement-receptor function in resident and thioglycolate-elicited mouse peritoneal macrophages. J. Exp. Med. 150, 607.

Phaire-Washington, L., Wang, E. and Silverstein, S. C. (1980).Phorbol myristate acetate stimulates pinocytosis and membranespreading in mouse peritoneal macrophages. J. Cell Biol. 86, 634.

Rablnovltch, M., Manejias, R. E. and Nussenzwelg, V. (1975).Selective phagocytic paralysis induced by immobilized immunecomplexes. J. Exp. Med. 142, 827-838.

Stelnman, R. M., Brodie, S. E. and Cohn, Z. A. (1976). Membraneflow during pinocytosis - a stereologic analysis. J. Cell Biol. 68, 665.

Swanson, J. (1989a). Fluorescent labeling of endocyticcompartments. In Fluorescence Microscopy of Living Cells inCulture. Methods in Cell Biology, vol. 29 (ed. Y. L. Wang and D. L.Taylor), pp. 137-151. Academic Press, NY.

Swanson, J. A. (1989b). Phorbol esters stimulate macropinocytosisand solute flow through macrophages. J. Cell Sci. 94, 135-142.

Swanson, J. A., Bushnell, A. and SUversteln, S. C. (1987). Tubularlysosome morphology and distribution with macrophages dependon the integrity of cytoplasmic microtubules. Proc. Nat. Acad. Sci.U.S.A. 84, 1921.

Swanson, J. A., Yirinec, B., Burke, E., Bushnell, A. and Silverstein,S. C. (1986). Effect of alterations in the size of the vacuolarcompartment on pinocytosis in J774.2 macrophages. J. Cell Biol.128, 195.

Takemura, R., Stenberg, P. E., Balnton, D. F. and Werb, Z. (1986).Rapid redistribution of clathrin onto macrophage plasmamembranes in response to Fc-receptor-ligand interaction duringfrustrated phagocytosis. / Cell Biol. 102, 55-69.

Wright, S. D. and SUverstein, S. C. (1982). Tumor-promoting phorbolesters stimulate C3b and O b i receptor-mediated phagocytosis incultured human monocytes. J. Exp. Med. 156, 1149-1164.

(Received 9 October 1991 - Accepted 17 December 1991)