907
Journal of Cell Science 101, 907-913 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
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, which
measure 13.8 ± 2.3 /an diameter in suspension, can
ingest IgG-opsonized latex beads greater than 20 /on
diameter. A precise assay has allowed the determination
of the phagocytic capacity, and of physiological parameters that limit that capacity. Ingestion of beads larger
than 15 /mi diameter required IgG-opsonization, and
took 30 minutes to reach completion. Despite the
dependence on Fc-receptors for phagocytosis of larger
beads, cells reached then* limit before all cell surface Fcreceptors were occupied. The maximal membrane
surface area after frustrated phagocytosis of opsonized
coverslips was similar to the membrane surface area
required to engulf particles at the limiting diameter,
indicating that the capacity was independent of particle
shape. Vacuolation of the lysosomal compartment with
sucrose, which expanded endocytic compartments,
lowered the phagocytic capacity. This decrease was
reversed when sucrose vacuoles were collapsed by
incubation of cells with invertase. These experiments
indicate that the phagocytic capacity is limited by the
amount of available membrane, rather than by the
availability of Fc-receptors. The capacity was also
reduced by depolymerization of cytoplasmic microtubules with nocodazole. Nocodazole did not affect the
area of maximal cell spreading during frustrated
phagocytosis, but did alter the shape of the spread cells.
Thus, microtubules may coordinate cytoplasm for
engulfment of the largest particles.
Introduction
macrophage could engulf under various conditions
allowed for the examination of several interesting
characteristics of phagocytosis. Cell surface area at the
phagocytic limit was similar to that reached after
frustrated phagocytosis. Fc-mediated phagocytosis
stopped before all Fc-receptors were engaged or
internalized, indicating that cell surface membrane
became limiting. Moreover, the phagocytic capacity
was lowered when microtubules were depolymerized.
Comparisons with frustrated phagocytosis suggested
that the radial symmetry of the phagocytic response
enhanced the phagocytic capacity.
Macrophages can ingest great quantities of particles,
yet not so much that they burst. When plated onto flat
surfaces opsonized with IgG, they engage that surface
as if to engulf it, a process termed frustrated phagocytosis (Henson, 1971; Takemura et al., 1986), and spread
to some limit. It may be that the macrophage capacity
for phagocytosis or its extent of spreading during
frustrated phagocytosis is limited by the number of
available phagocytic receptors, and when all of them
have been internalized or engaged phagocytosis stops.
Alternatively, some cellular structure which changes
during the process may approach a limit, and that limit
defines satiety.
The goals of the present experiments were to
determine the phagocytic capacity of murine bone
marrow-derived macrophages, to compare that capacity
to frustrated phagocytosis, and to identify factors that
limit those capacities. Capacity was measured using
polystyrene beads that range in size from those that
could easily be ingested by one cell to those that were
significantly larger than the cell itself. The cell and bead
concentrations were adjusted so that the usual condition consisted of one cell attempting to phagocytose
one bead. Determining the largest bead that the
Key words: macrophage, phagocytosis, Fc-receptors,
microtubules.
Materials and methods
Cells
Murine bone marrow-derived macrophages were obtained by
the method of Swanson (1989b). Bone marrow was removed
from femurs of female ICR mice (Charles River, Cambridge,
MA) and was washed 3x in cold Dulbecco's modified
essential medium plus 10% heat-inactivated fetal bovine
serum (DME-10F). Cells were resuspended and plated at 2 X
105 cells/ml on 100 mm Lab-Tek dishes in 25 ml or on 60 mm
Lab-Tek dishes in 10 ml of complete bone marrow medium
(DME plus 30% L-cell-conditioned medium plus 20% heatinactivated 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 adhered
to the bottom of the dish during this period. Macrophages
were 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 in
24-well Costar dishes at 1 x 105 cells/ml. After 30 minutes at
37°C, the PD was replaced with DME-10F. Cells were used
within the next two days. Greater than 95% of the cells were
macrophages, as determined by their ability to phagocytose
opsonized sheep red cells.
Macrophages resuspended from Lab-Tek dishes into cold
PD were examined microscopically (magnification, X500).
Diameters of 83 rounded, unspread cells were measured using
a calibrated eyepiece graticle.
Beads
2 x 106 polystyrene polybeads of diameter 21.1 fjm (Polysciences Inc., Warrington, PA) were incubated in 10 mg/ml
bovine serum albumin (BSA, Sigma Chem. Co., St. Louis) in
PD for 1 hour at 4CC. The beads were then washed 5 times in
PD, then resuspended in 1 ml PD. Rabbit anti-bovine
albumin, IgG fraction (anti-BSA IgG, Cappel, West Chester,
PA), was added to a final dilution of 1:500, then incubated for
30 minutes at 37°C and 10 minutes at 4°C. The beads were
then washed 3 x in 1 ml PD and recounted. The 21.1 jum
beads had a considerable variance in size (4.09 /an, according
to the manufacturer) with a range of 13 jjm to >30 fim in
diameter, and this made the phagocytosis assay possible.
For other phagocytosis assays, 21.1 /m\ BSA-coated beads
were incubated in 6 mg/ml 2,4-dinitrobenzene sulfonate
(DNBS, Aldrich Chem. Co., Milwaukee), in 4% K2CO3 for 1
hour, then washed 3 x in 1 ml PD. The beads were then
incubated in anti-DNP IgG before or after they came in
contact with the macrophages.
Basic phagocytosis assay
A quantitative measurement for the phagocytic capacity of
macrophages was obtained by allowing the cells to engulf
large latex beads, then using immunofluorescence to label
beads that were not internalized, and measuring how many
beads of a given size interval were positively labeled.
Fresh DME-10F (1 ml) was added to day 7 or day 8
macrophages. 2 x Mr beads (21.1 fan diameter) were added
to each well. Cells were allowed to phagocytose the beads for
one hour. Dishes were then placed on ice, washed gently 3 x
in cold PD, the wells aspirated dry, and rhodamine-labeled
goat anti-rabbit IgG (rhodamine-GAR: heavy and light chain
specific, Cappel), 300 /A at 1:50 in PD, was added. The cells
were then incubated on ice for 30 minutes and washed 3 x in
PD. Control experiments showed that only beads which had
both BSA and anti-BSA on them could be labeled with the
rhodamine-labeled secondary antibody (data not shown).
Even though 30 minutes at 4°C was allowed for the
rhodamine-labeling step of the assay, beads were completely
labeled 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 prevent
drying (Swanson, 1989a). The cells were studied with a Zeiss
Photomicroscope i n equipped for epi-illumination of fluorescent specimens. To measure bead diameters, a Wert xlO
ocular micrometer was calibrated with a stage micrometer.
The divisions on the ocular micrometer were 1.8 micrometers
apart at x500. For each bead, the diameter was measured and
the bead scored as plus or minus for rhodamine fluorescence
with rhodamine filters. Only in cases where there was one
bead per macrophage, and one macrophage per bead, were
measurements taken. Although a phase 3 lens was used to
view the cells, a condenser lens without phase rings was used
during bead diameter measurement to avoid optical distortion
of bead dimensions. The diameter of the beads could be
measured to the nearest micrometer with considerable
accuracy. For each 1.8 /.an gradation, 10 beads were scored for
fluorescence.
Determination of the parameters of phagocytosis
All of the experiments described in this section were executed
in their entirety at least twice.
Time course of phagocytosis
The beads were assayed as above, except that phagocytosis
was allowed to proceed for shorter periods of time: 5,10, 20,
30 and 60 minutes. Coverslips were removed from the dish
and placed in a new dish on ice with 1 ml PD in each well.
Rhodamine-GAR was added after the cells were chilled and
washed.
Phagocytosis of opsonized beads
BSA/anti-BSA-coated beads, BSA/DNP-coated beads (no
anti-DNP), and BSA/DNP beads that had been opsonized
with rabbit anti-DNP IgG were added to two wells. After one
hour of phagocytosis, anti-DNP was added to the unopsonized bead wells (300 /A of 50 j<g/ml in PD) for 30 minutes on
ice, while the others were kept in PD. After 3 washes, all were
incubated in the rhodamine-GAR.
Effect of nocodazole on phagocytic capacity
Macrophages were incubated in 10 JJM nocodazole (Sigma
Chemical, St. Louis, MO ) in DME-10F for one hour before
21.1 /un beads were added. The phagocytosis and labeling
procedures remained the same.
Effect of cell vacuolation
Macrophages were incubated overnight (15 hours) in 10
mg/ml or 20 mg/ml sucrose in DME-10F according to Swanson
et al. (1986). Phagocytosis and labeling protocols were
unchanged. For invertase recovery, cells that had been
incubating in 20 mg/ml sucrose for 15 hours were washed twice
in medium, then 1 ml of 0.5 mg/ml invertase (Sigma Chem.
Co.) in medium was added for 2, 4 or 6 hours before the
phagocytosis assay was performed. In control experiments,
vacuolated cells were maintained in sucrose for 4 hours before
allowing phagocytosis.
Macrophage spreading on opsonized coverslips
The extent of cellular spreading of macrophages that engage
in frustrated phagocytosis was determined. 12 mm coverslips
were treated with DNP according to the method of Michl et
al. (1979). Coverslips were opsonized with 50 /ig/ml anti-DNP
IgG in PD for 30 minutes and then washed 3 x in PD.
Macrophages that had been chilled and resuspended from
Lab-Tek dishes were plated onto the coverslips at 1-2 x
104/well. After 1 hour of spreading, the cells were chilled on
ice and then incubated with 300 [A rhodamine-GAR (1:100)
for 30 minutes to label areas of the coverslip which were not
masked by the macrophages. The cell diameters were
measured with the ocular micrometer.
For other studies, coverslips coated with BSA were
opsonized with anti-BSA IgG for 30 minutes, then washed
thoroughly before plating macrophages. Macrophages resuspended from Lab-Tek dishes were plated onto coverslips in
Ringer's saline ± 10 /JM nocodazole.
The macrophage capacity for phagocytosis
909
Erythrocyte binding to macrophages
Sheep red blood cells (SRBC: Cappel) were opsonized with
goat anti-SRBC IgG (Cappel) at a final dilution of 1:1000 (30
minutes 37°C, 10 minutes 4°C, washed 3 X in PD).
Macrophages that had phagocytosed test beads were incubated for 1 hour with 3 x 106 opsonized SRBC. Cells were
subsequently chilled and incubated in rhodamine-GAR. In
separate experiments, opsonized SRBC were added to
macrophages 1 hour after plating onto opsonized coverslips
(DNP-anti DNP). SRBC were incubated with the macrophages for 15 minutes at 4°C, then unbound SRBC were
washed away. The number of red cells on the surface of 100
randomly selected macrophages was measured.
Measurements of macrophage area and shape
Macrophages were allowed to spread for 40 minutes onto
coverslips 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, then
were washed and observed by phase-contrast microscopy.
Video images of 35 cells for each condition were recorded and
digitized for image processing. Outlines of cells were traced
using interactive software; these tracings were used to
generate a binary mask for each cell, then these binaries were
analyzed to measure area (A), perimeter (P) and the shape
parameter (P2/4;rA) for each cell using the image processing
capabilities of a Tracor Northern TN 8500 system (Noran
Inst., Middletown, WI).
Results
Phagocytic capacity
The phagocytosis assay measured the percentage of
latex beads within a given size range that could be
engulfed by macrophages. Only instances in which one
macrophage engaged one bead were acceptable for
measurements. Internalized beads were identified as
such by their inaccessibility to fluorescently labeled
antibodies against their surfaces. Beads bound to cells
were almost always completely dark or brightly fluorescent. Exceptions occurred when beads were partially
engulfed and thus only partially labeled; such beads
were scored positive for fluorescence (i.e. they had
failed to engulf the bead). The basic assay to determine
the phagocytic capacity of bone marrow-derived macrophages was performed as a separate experiment and as
a control for each of the subsequent experimental
variations. The data were averaged and expressed as
the percentage of positively labeled beads in each bead
size category (Fig. 1). An operative definition for the
phagocytic limit was given as the bead diameter at
which 50 per cent of the beads scored positive for
rhodamine labeling. In order to determine this point, a
logit transformation was performed on the data to make
the sigmoidal curve linear, and then first-order regression analysis was performed. In the control condition, the 50% mark occurred at 19.8 /im, with a 95%
confidence interval of 17.0 jim to 22.5 ^urn. Macrophages
resuspended from a culture dish assume a spherical
morphology. The diameters of resuspended macrophages were found to be 13.8 ± 2.3 /xm (s.d.). The
calculated phagocytic capacity of 19.8 fim indicates that
r4
18
20
Bead diameter (/mi)
22
24
Fig. 1. Determination of the macrophage capacity for
phagocytosis. A mixture of opsonized particles of varying
diameter was added to macrophages. After 60 min for
phagocytosis, the cells were chilled and incubated with
rhodamine-labelled goat anti-rabbit IgG, to decorate
exposed particle surfaces. Ten beads of each size class, that
also contained an associated macrophage, were scored as
positive (external) or negative (internalized) for rhodamine
fluorescence. Data from several experiments were pooled
to obtain the values shown. The capacity was operationally
defined as the bead diameter at which half the beads were
engulfed. The schematic drawings indicate the principle of
the labelling procedure, with bristles indicating the exposed
surface of the larger particle (left).
14
16
18
20
Bead 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 before
chilling and labelling with fluorescent secondary antibodies.
Cells reach their final capacity with 30 minutes of
phagocytosis.
those cells are capable of ingesting particles 1.44 times
their diameter, or 3 times their volume.
The time course of phagocytosis was determined by
varying the time that the macrophages were exposed to
the beads at 37°C. As Fig. 2 indicates, macrophages
reached 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 at
20 minutes was already not significantly different from
the control values (50%=18.4 jm\, P<0.40).
To examine the role of opsonization in the phagocytosis of larger beads, BSA-coated beads were labeled
with dinitrobenzene sulfonate (DNBS) before incubation with the cells, and phagocytosis was measured by
chilling and labelling the beads with anti-DNP and then
rhodamine-GAR. Macrophages phagocytosed DNP/
anti-DNP coated beads (50% = 19.1 fzm, P>0.50) to the
same 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 receptorligand interactions as necessary for the phagocytosis of
larger beads.
Measurements of cellular spreading
We compared the phagocytic capacity for opsonized
beads with the extent of macrophage spreading on
opsonized substrata. This frustrated phagocytosis
(Henson, 1971; Michl et al., 1979; Takemura et al.,
1986) is analogous to the phagocytosis of an infinitely
large particle. A significant difference in values obtained for phagocytosis of beads versus those for
spreading would indicate that the cells can distinguish
shapes of particles during phagocytosis. Due to the
tightness of the seal that the spread cells formed with
the coverslip, and the fact that cells that were not
touching any other cells would spread into circular
profiles (Fig. 3A), a simple measurement of the
diameter of the circular profile permitted estimation of
cell spreading and surface area. Since cells either
tended to clump together in suspension, or to contact
each other as they spread, only the 10 largest, isolated
and circular macrophage profiles on each coverslip were
measured. The average diameter of the 10 largest cells
on coverslips from several different cultures of macrophages 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 of
the cell, the area should be doubled to yield a cell
surface area of 3,080 fim2. This estimate is a minimum
value for the surface area, since it considers the cell as
an infinitely thin disk. It is a fair estimate, however,
since cell ruffling is considerably reduced in this
condition, and the cells are very flat (Fig. 3A).
Moreover, our estimate is in good agreement with other
determinations of surface area obtained in thioglycolate-elicited peritoneal macrophages (Phaire-Washington, 1980). This limit is similar to the surface area
required to ingest the largest beads. To engulf a bead of
19.8 ;Um, the macrophage plasma membrane must reach
a surface area of at least 2,463 ^m2 (2 x 1232 ^im2). This
similarity between bead phagocytosis and frustrated
phagocytosis indicates that at their limit these two
processes attain the same total cell surface area, and
that macrophages do not discriminate particle shapes in
these processes. The difference of 600 (im2 may be
explained by the difference in the way the limits are
defined. For frustrated phagocytosis we measured the
10 largest circular cell profiles, essentially identifying
Fig. 3. Nocodazole treatment does not inhibit frustrated
phagocytosis, but alters the symmetry of macrophage
spreading. Macrophages were plated onto opsonized
coverslips for 40 min with (B) or without (A) 10 f.an
nocodazole, then were fixed for microscopy. Bar, 20 fan.
champions, whereas for bead phagocytosis we attained
the half maximal bead size for phagocytosis, which
measures the capacities of the whole population. The
latter method would be expected to yield lower
numbers.
Fc receptors on the surfaces of the macrophage
Although it appears that membrane availability is the
limiting factor with respect to phagocytic capacity,
another reason why the cells might not be able to ingest
more could be that they deplete from their surfaces all
receptors for binding with the opsonized particles. To
test this, opsonized sheep erythrocytes were added to
either normally spread macrophages that had phagocytosed a large bead or macrophages that were
engaging a coverslip in frustrated phagocytosis. As
shown in Fig. 4, macrophages spread onto opsonized
surfaces could bind red cells on their upper surface. A
The macrophage capacity for phagocytosis
Fig. 4. Frustrated phagocytosis does not deplete the cell
surface of Fc-receptors. Macrophages were allowed to
engage the opsonized coverslip for 60 min, then were
chilled and provided opsonized erythrocytes. Macrophages
continued to bind erythrocytes even after spreading fully.
count of erythrocytes per 100 cells yielded 15 (±6)
bound erythrocytes per macrophage. Similarly, macrophages engaging large, opsonized beads could also bind
opsonized erythrocytes (data not shown). These results
indicate that the phagocytic capacity is not limited by
the availability of receptors.
Role of internal compartments
The cell surface areas reached by phagocytosis were
considerably greater than the reported macrophage
membrane surface area (825 ^ m ; Steinman et al.,
1976), indicating that internal membranes are recruited
to the plasma membrane during phagocytosis. We
asked how expanding internal membranous compartments affected the engulfment of larger beads. To do
this, the endocytic and lysosomal compartments were
vacuolated during a 15 hour incubation in sucrose.
Macrophages lack invertase, so pinocytosed sucrose
accumulates in lysosomes, where it expands that
compartment osmotically (Cohn and Ehrenreich,
1969). Cells were incubated overnight in various
concentrations of sucrose and then the phagocytosis
assay was performed (Table 1). The ability of macrophages to ingest beads was drastically reduced after
Table 1. Phagocytic indices following vacuolation with
sucrose or invertase-mediated sucrose vacuole collapse
overnight vacuolation in 30 mg/ml and 20 mg/ml
sucrose. 10 mg/ml sucrose also had an appreciable
effect, reducing the 50% labeled value to 16.4 urn
(P<0.02).
To determine if this decrease in the size limit was not
due to general impairment of cells, the vacuolation was
reversed with invertase. Invertase degrades sucrose to
its component monosaccharides, which exit lysosomes
and permit shrinkage of the lysosomal compartment
(Cohn and Ehrenreich, 1969; Knapp and Swanson,
1990; Swanson et al., 1986). Cells were vacuolated in
sucrose for 15 hours and then washed in medium. They
were then incubated in 0.5 mg/ml invertase for up to 6
hours (Table 1). Within 2 hours the phagocytic capacity
had nearly returned to pre-vacuolation levels. A small
increase was seen at the 4 hour and 6 hour time points.
The invertase-treated cells approached but did not
reach pre-vacuolation levels.
Phagocytosis without microtubules
To determine if microtubules were necessary for
phagocytosis of the beads, cells were incubated in 10
L*M nocodazole, which causes reversible depolymerization of macrophage microtubules (Swanson et al.,
1987), for 1 hour before and during incubation with the
beads. Nocodazole produced a small but significant
decrease in the amount the cells could ingest (50% =
16.9 Mm, P<0.05).
Because depolymerization of cytoplasmic microtubules reduced the phagocytic capacity, we compared
this response to frustrated phagocytosis and found that
nocodazole did not limit the extent of spreading, but
instead made that spreading response irregular.
Whereas the usual frustrated phagocytic response
created circular spreading profiles, with the nucleus
centrally placed, macrophages spreading in nocodazole
formed more irregular shapes (Fig. 3). We quantified
these observations by tracing the profiles of cells spread
onto opsonized coverslips for forty minutes in the
presence or absence of 10 /*M nocodazole. Using digital
image processing, we determined spreading area and
the deviation of the profile from a circle (Table 2). Cells
were not significantly different in spread areas but were
significantly different in shape. This indicates that
phagocytosis of the largest particles is enhanced by a
microtubule-dependent organization of cytoplasm.
Table 2. Macrophage area and shape after frustrated
phagocytosis
Profile
Condition of cells
Sucrose (mg/ml)
in overnight
incubation
10
20
30
20
20
20
911
Invertase
treatment
(h)
2
4
6
50% Phagocytosis
[an
P
16.4
14.9
<14.0
17.6
18.4
18.7
(<0.02)
(<0.001)
(<0.001)
(<0.10)
(<0.20)
(<0.40)
Cells
Control
Nocodazole
Area
Shape
844<5±552
9382±952
1.28±0.30
2.25±0.26
Area and shape are determined from binary images, obtained by
interactive image processing. Areas are reported as number of
pixels per cell. Shape is expressed as perimeter (P) squared,
divided by 4JI times the area (A). (P 2 /4^A). A circle would have a
shape index of 1.0.
912
G. J. Cannon and J. A. Swanson
Discussion
Measurements using the phagocytic assay described
here indicate that frustrated phagocytosis and particle
phagocytosis are limited in similar ways, but are
different from cell spreading onto unopsonized surfaces. Grinnell and Geiger (1986) described the phagocytosis by fibroblasts of various sizes of fibronectincoated beads and found that fibroblasts could phagocytose 6 /xm diameter beads easily, but could not engulf
beads larger than 12-14 /an diameter. In the light of
earlier work in which prior spreading on fibronectincoated surfaces inhibited phagocytosis of fibronectincoated beads (Grinnell, 1980), they concluded that
phagocytosis and cell spreading on flat surfaces were
fundamentally the same process, which was limited
either by the number of fibronectin receptors or by
some other physical constraint.
We found that particle capacity was increased by
opsonization, but was not limited by Fc-receptor
availability. Macrophages at their phagocytic capacity,
either with a bead inside or spread to their limit,
remained capable of binding opsonized erythrocytes on
their upper surfaces. The limit instead appeared to be in
the amount of cell surface membrane available for
spreading. Macrophages in suspension, or plated onto
unopsonized surfaces, contain prominent ruffles of
membrane. When engaging an opsonized surface,
ruffling membrane moves along that surface and
flattens against it. As the limit is approached, surplus
membrane is recruited into the phagocytic response,
and the cell surface is drawn smooth (Fig. 5). The limit
seems therefore set by the amount of available
membrane. An alternative explanation has been
offered by Rabinovitch et al. (1975), that frustrated
phagocytosis is not limited by spreading, but rather by a
paralysis of Fc-receptors for phagocytosis. In their
studies, murine peritoneal macrophages plated onto
opsonized coverslips could bind but not ingest opsonized erythrocytes. They argued that this inhibition was
not a consequence of cell spreading because other
conditions which increased cell spreading did not inhibit
phagocytosis. It therefore remains possible that macrophage frustrated phagocytosis is limited by a specific
inactivation of Fc-receptor function, an inhibition
mediated by receptor ligation.
Our estimates of the cell surface area at capacity are
similar for both bead phagocytosis and frustrated
phagocytosis, and are also similar to values reported in
earlier studies of phorbol ester-stimulated spreading
(Phaire-Washington et al., 1980). Those surface areas
are greater than those measured in unstimulated
macrophages (825 /zm2; Steinman et al., 1976), indicating that intracellular membranes are brought to the
surface during phagocytosis. Occupation of internal
membranes, such as occurred during sucrose vacuolation, decreased the phagocytic capacity reversibly,
indicating that these membranes contribute to the
spreading. It remains possible, however, that macrophage surface area does not change during phagocytosis; that the highly folded surface simply smooths out to
Fig. 5. The extent of cytoplasmic reorganization during
phagocytosis of the largest particles or during frustrated
phagocytosis. (A) The macrophage in suspension has a
mean diameter of 14 fan, and an extensively ruffled
surface. Macrophages at their limits of particle
phagocytosis (B) or frustrated phagocytosis (C) are much
larger than unfed cells, and contain very few surface
ruffles. The phagocytic capacity may therefore be limited
by the amount of available cell surface membrane.
enclose particles, and that filling endocytic compartments with sucrose or smaller particles depletes the cell
surface of membrane which would otherwise be
available for the spreading response.
The similar limits of particle phagocytosis and
frustrated phagocytosis indicate that they are not
affected by particle shape or by the size of the
phagosomal lamellipod. The advancing edge of the
pseudopod decreases beyond the equator of a spherical
particle, but continually increases on a flat surface.
Pseudopod advance therefore appears to be regulated
locally, by the segmental interactions between Fcreceptors and the opsonized surface, and with the limit
of advance set by the amount of available surface
membrane.
That both particle phagocytosis and frustrated phagocytosis proceed in the absence of cytoplasmic microtubules suggests that the process is independent of this
cytoskeletal element. Macrophages plated onto opsonized surfaces in the presence of nocodazole engage
those surfaces in a phagocytic response, and reach the
same spreading surface area as control cells. However,
the phagocytic capacity for particles is diminished by
nocodazole treatment, indicating that microtubules
contribute to particle phagocytosis in some way. We
suggest that microtubules provide a coordinating func-
The macrophage capacity for phagocytosis
tion. Such coordination is indicated by the striking
symmetry of cell spreading during frustrated phagocytosis. Macrophages spread into nearly perfect circles,
with the nucleus centrally placed and with microtubules
radiating to the cell periphery from a central point. In
the absence of microtubules, such spreading may be
initially circular, but soon rearranges to an irregular
profile. The radial symmetry that is lost during
nocodazole treatment may reflect a process that
coordinates pseudopod closure around large particles.
Without microtubules, uncoordinated pseudopod advance around particles would produce irregularly
shaped gaps that close less readily than the small,
circular gaps left by symmetrical spreading.
In contrast to Fc-mediated phagocytosis (Bhisey and
Freed, 1971), complement-mediated phagocytosis in
macrophages is sensitive to microtubule-destabilizing
drugs (Wright and Silverstein, 1982). Complementmediated phagocytosis therefore may be more similar
to simple spreading processes, which are sensitive to
nocodazole, than to Fc-mediated phagocytosis.
Measurements of the phagocytic capacity of complement-opsonized particles might be instructive in this
regard.
The authors gratefully acknowledge the assistance and
advice of Philip Knapp and Esther Racoosin. This work was
supported by the NIH (CA44328).
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(Received 9 October 1991 - Accepted 17 December 1991)