EXPERIMENTAL CELL RESEARCH 223, 426–435 (1996)
ARTICLE NO. 0098
Guidance and Activation of Murine Macrophages
by Nanometric Scale Topography
BEATA WÓJCIAK-STOTHARD,*,1 ADAM CURTIS,* WILLIAM MONAGHAN,†
KAREN MACDONALD,* AND CHRIS WILKINSON†
*Laboratory of Cell Biology and †Department of Electronics and Electrical Engineering, University of Glasgow,
Glasgow, G128QQ, Scotland, United Kingdom
We studied the guidance and activation of macro-
phages from the P388D1 cell line and rat peritoneum
by topographic features on a nanometric scale. Cells
were plated on plain fused silica substrata or substrata
with microfabricated grooves and steps, 30–282 nm
deep. The contact of cells with the patterned surface
activated cell spreading and adhesion and increased
the number of protrusions of the cell membrane. These
changes were accompanied by an increase in the
amount of F-actin in cells. The accumulation of F-actin
and vinculin in cells was observed along the edges of
single steps or grooves. Formation of focal contacts
along discontinuities in the substratum was accompa-
nied by the phosphorylation of tyrosine colocalized
with F-actin and vinculin. A similar pattern of staining
was seen in cells stained for vitronectin receptor, aV
integrin, but not for integrins a5b1 or a3b1. Cells cul-
tured on nanogrooves showed a higher phagocytotic
activity than cells cultured on plain substrata. We
show that macrophages can react to ultrafine features
of topography of a size comparable to that of a single
collagen fiber and become activated by the contact
with topographic features. q 1996 Academic Press, Inc.
INTRODUCTION
It has been shown in a number of studies that cells
can be guided by oriented fibrils of an extracellular
material [1–3]. Since the cell environment in vivo pro-
vides a variety of mechanical, chemical, and topograph-
ical stimuli, the mechanism of cell guidance is difficult
to assess. Chemotaxis was believed to be the leading
force of directional cell movement for the cells of the
immunological system [4]. It has been shown, however,
that topography can enhance the chemotactic response
1
To whom reprint requests should be addressed at Laboratory of
Cell Biology, Division of Molecular and Cellular Biology, Glasgow
University, G128QQ Glasgow, Scotland, UK. Fax: 0141-330-4501; E-
mail: gbaa10@udcf.gla.ac.uk.
0014-4827/96 $18.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
/ 6i0a$$3079 02-13-96 23:28:40 ecas
of human neutrophil leukocytes [3]. Multiple grooved
substrata have been used by several authors as a model
of oriented topographical features of the extracellular
matrix [5–8] but the size of grooves was much greater
than the size of matrix proteins. In this paper we use
multiple grooved substrata with a groove depth as
small as 30 nm, which corresponds to the diameter of
a single collagen fiber. It has recently been shown that
substrata with grooves of a greater size stimulate the
movement of a variety of cells [9–11] and increase the
amount of fibronectin secreted and assembled into the
extracellular matrix by human gingival fibroblasts
[25]. Murine macrophages have been shown to respond
to patterned surfaces by extensive spreading accompa-
nied by the increase in the persistence and speed of
cell movement [10]. In this paper we report the reaction
of murine macrophages from the P388D1 cell line and
rat peritoneum to a substratum which has features on
a nanometric scale. The contact of cells with patterned
substrata induces several changes in cell morphology
and activity and is accompanied by changes in the dis-
tribution and content of cytoskeletal components. Us-
ing these observations, possible mechanisms for the
reaction of cells to ultrafine topographic features are
discussed.
MATERIALS AND METHODS
Cells
The P388D1 macrophage cell line was a kind gift of Professor P.
Bongrand, Marseille. Cells were grown in RPMI 1640 medium
(Gibco, Life Technologies, Inc. Grand Island, NY) supplemented with
10% fetal calf serum (Sigma Chemical Co), 20 mM Hepes buffer
(N-2-hydroxyethylpiperazine-N*-2-ethanesulfonic acid, pH 7.4), 100
units/ml penicillin, and 10 mg/ml streptomycin at 377C. Cells were
grown in 25-ml TC Falcon flasks and passaged biweekly. Cells were
plated at the density 1 1 105 cells/ml onto grooved or plain substrata
and incubated at 377C. After 15 min of incubation cells were fixed
in 4% formaldehyde and stained with Coomassie blue. The cell area,
elongation, and orientation were then calculated with the use of
image analysis programs.
Rat macrophages were obtained by the peritoneal lavage of 6-week-
old Sprague–Dawley rats. Cells were suspended in phosphate-buf-
426
AP: Exp Cell
 REACTION OF MACROPHAGES TO NANOMETRIC SCALE TOPOGRAPHY
fered saline (PBS), centrifuged, resuspended in culture medium, and
plated onto plain and grooved substrata.
Substratum Patterning
For experiments we used fused silica substrata with micropat-
terned arrays of parallel grooves and ridges of equal width and a
square wave profile. Fused silica samples (Multilab, Newcastle, UK)
were cut into 25-mm2, 1-mm-thick samples. The silica was cleaned
by soaking in a solution of 3:1 sulphuric acid:hydrogen peroxide for
5–10 min at 607C followed by rinse in R.O. water and then blown dry
with filtered air. The silica was coated with AZ 1400-31 photoresist
(Shipley, Coventry, UK) by spinning-on at 4000 rpm for 30 s followed
by a soft bake at 907C for 30 min. This gave a resist thickness of 1.8
mm. The resist was then patterned by exposing to UV light for 10 s
through a chrome mask patterned with the required grating pattern,
using a mask aligner (HTG, San Jose, CA). The exposed resist was
removed by immersing the sample in a solution of 1:1 Shipley devel-
oper: R.O. water for 65–75 s followed by a rinse in R.O. water and
blown dry. The samples were dry etched in a Reactive Ion Etching
Unit (RIE80, Plasma Technology, Bristol, UK) at a rate of approxi-
mately 30 nm/min. One hundred watts of rf power at a frequency of
13.6 MHz powered a 15-cm-diameter table; CHF3 was used at a
flow rate of 20 sccm and a pressure of 15 mT. The self-bias was
approximately 0480 V. After etching, the residual resist was re-
moved in warm acetone, and all samples were etched for an addi-
tional minute without protection to ensure uniform surface chem-
istry.
We used both plain fused silica substrata and coated fused silica
substrata. In general the substrata were used directly after cleaning
without addition of a protein layer. However, in some cases substrata
were incubated with 200 ml of 10 mg/ml fibronectin or 1 mg/ml conca-
navalin A for 1 h at room temperature. Substrata covered with ConA
were left to air dry and then all coated surfaces were washed five
times in PBS.
The depth of grooves was measured with 5% accuracy with DEK-
TAK Surface Profiler (Viico Sloan Technology Division, Santa Bar-
bara, CA).
Image Analysis System: Measurement of Cell Area, Orientation,
and Adhesion
Images from a Leitz Ortholux microscope using a Leitz Phaco 10X
objective were digitized to 512 1 256 pixels and 64 gray shades with
a Hamamatsu Vidicon C1000 camera (Hamamatsu c/o. Nikon UK,
London) and analyzed with an Archimedes digitizer (Watford Elec-
tronics, Watford, UK). The image analysis program, written by Chris
Edwards (Chemistry Department, Glasgow University) in Acorn Risc
machine assembler language for an Acorn Archimedes 310 microcom-
puter, was used to calculate the number of cells/field and measure
the area of the cells and their orientation. These parameters were
calculated as described by Dunn and Brown [12].
Fluorescent Staining and the Measurement of F-actin Content
in Cells
Fifteen minutes after plating, cells on plain and patterned struc-
tures were rinsed in PBS, fixed in 1% glutaraldehyde for 15 min,
and then permeabilized in 0.5% Triton X-100 for 15 min. After drain-
ing, each structure was incubated in 1 mg ml01 TRITC-phalloidin
solution in PBS for 45 min and then washed twice in PBS and exam-
ined under the Leitz fluorescent microscope. The brightness of fluo-
rescence (integrated brightness) of each individual cell, proportional
to F-actin content, was measured with the MetaMorph Imaging Sys-
tem (Universal Imaging Corporation, West Chester, PA). The fluo-
rescence of 50 cells was measured on each kind of substratum.
/ 6i0a$$3079 02-13-96 23:28:40 ecas
427
Fluorescent Staining of Vinculin, ICAM-1, Integrins a5b1, aVb1,
a3b1, and Phosphotyrosine
Cells were washed in PBS (prewarmed to 377C), fixed in 4% formal-
dehyde, and then permeabilized in 0.5% Triton X-100 for 15 min.
After draining, each coverslip was incubated with 200 ml of the follow-
ing antibodies: mouse monoclonal anti-human vinculin antibody
(Sigma) 1:100; mouse monoclonal anti-rat ICAM-1 (Serotech Ltd.,
Kidlingtom, Oxford, England), 1:200; mouse monoclonal anti-phos-
photyrosine (PY20) (ICN Pharmaceuticals, Thame, England) for 2 h
at room temperature.
To stain for integrins, the cells were fixed in 0.25% glutaraldehyde
for 15 min and then washed three times in PBS and incubated with
a 20 mM solution of sodium borohydride in PBS for 1 h. Fixed cells
were permeabilized in 0.1% Triton X-100 in PBS for 30 min. Cells
were incubated with 200 ml of the following antibodies: mouse mono-
clonal anti-a5b1 integrin (Gibco), rabbit polyclonal anti-a3 integrin
(Chemicon, Harrow, England) 1:100, or rabbit polyclonal anti-aV
integrin (Chemicon) at a dilution 1:100. Then cells were washed three
times in PBS and incubated with 200 ml of the secondary antibodies:
sheep Texas red-linked anti-mouse antibody (Amersham Life Sci.)
or donkey Texas red-linked anti-rabbit antibody (Amersham Life
Sci.) 1:100 for 2 h and washed three times in PBS. None of the
controls with fixed cells incubated either with the first or with the
second antibody alone showed positive staining.
Interference-Reflection Microscopy and Confocal Laser
Scanning Microscopy
P388D1 macrophages cultured on nanogrooved substrata for 15
min were examined under the interference-reflection microscope at
377C using a 100X objective.
Cells stained for fluorescence were examined under the Odyssey
Real Time Laser Scanning Microscope Model No. VSM-LSM (Noran
Instruments, Inc., Milton Keynes, UK) equipped with an argon-ion
laser, working with the MetaMorph Imaging System (Universal Im-
aging Corporation). Cells stained for actin were examined under 529
nm excitation wavelength with a 15-mm slit 60X objective.
Transmission Electron Microscopy (TEM)
Cells on quartz coverslips were fixed in 2.5% glutaraldehyde in 0.1
M phosphate buffer, pH 7.4, for 1 h. After fixation, cells were rinsed
in 0.1 M phosphate buffer with 2% sucrose and incubated with 1%
w/v osmium tetroxide at 377C for 1 h at room temperature, washed
in distilled water, and then incubated in 0.5% aqueous uranyl acetate
for 1 h in the dark. Samples were then rinsed in distilled water and
dehydrated by incubation in 30, 50, and 70% and absolute alcohol
for 10 min in each concentration. Cells were transferred to propylene
oxide and changed three times for 5 min and finally left in a 1:1
mix of propylene oxide:Araldite for 30 min. Cells were embedded in
Araldite at 607C for 48 h to polymerize. Polymerized resin and cov-
erslip with gratings were dipped in liquid nitrogen to separate the
coverslip from the cells and resin. The Araldite block was embedded
cell side down again on fresh resin and polymerized for another 48
h. The block was sectioned on a Reichert ultramicrotome and sections
were stained with 2% methanolic uranyl acetate for 5 min and in
Reynolds lead citrate for 5 min. A range of thicknesses of sections
(60–150 nm) were viewed on a Zeiss 902 transmission electron micro-
scope using zero energy loss filtering.
Phagocytosis Assay
To determine the phagocytotic activity of cells we used the method
of Van Furth [13]. FITC-labeled polystyrene beads (Polysciences Flu-
oresbrite Microspheres), 0.94 mm in diameter, were suspended in
culture medium at a density of 4.5 1 107 beads/ml. Cells were incu-
AP: Exp Cell
428 WÓJCIAK-STOTHARD ET AL.

FIG. 1. P388D1 macrophage spread on (a) plain fused silica substratum. (bar, 5 mm); (b) well-spread macrophage with numerous
microvilli on microgrooved substratum, groove depth 71 nm. (bar, 5 mm); (c) P388D1 macrophages spread on plain (rhs) and grooved (lhs)
substratum, 15 min after cell plating (bar, 20 mm). (a) and (b) represent scanning electron micrographs, (c) is a phase-contrast image.

bated with fluorescent bead suspension for 5 h at 377C. Then slides
were rinsed thoroughly with PBS to remove the excess of beads, and
cells were fixed with methanol for 10 min and allowed to air dry.
Slides were then flooded with xylene, air dried, mounted in 50%
glycerol, and examined under a fluorescent microscope. The number
of beads/cell was counted in 200 cells on each kind of substratum.
Statistical Analysis
For each variation of substrate and treatment, 100 cells were ana-
lyzed. Means, standard deviations, and variances were calculated
for the cell spreading area, elongation, orientation, and the parame-
ters of cell movement. Student’s t test was used to compare groups
of results.
RESULTS
Changes in Cell Spreading, Morphology, and
Orientation
P388D1 macrophages cultured on plain fused silica
substrata attached and spread in 20–30 min (Fig. 1).
Fifteen minutes after plating most cells remained
attached but rounded and not very well spread. As re-
vealed by the electron scanning micrographs, most of
the cells had fairly smooth surface with only few mi-
crospikes (Fig. 1a). The average cell spread area was
430 mm2, SD 160.
Cells plated on patterned substrata had a substan-
tially different morphology from cells on plain sub-
strata (Figs. 1b and 1c). Fifteen minutes after plating
cells were already attached and well spread. Cells ex-
tended numerous microspikes, usually perpendicular
to the groove direction (Fig. 1b). The spread area of
cells on 282-nm-deep grooves was two times greater
than the spread area of cells on a plain surface. The
reaction of cells to nanogrooves continued for as long
as the cells were cultured. Table 1 shows data on
spread areas and orientation of P388D1 macrophages
and normal rat peritoneal macrophages on grooves 30–

/ 6i0a$$3079 02-13-96 23:28:40 ecas AP: Exp Cell

 REACTION OF MACROPHAGES TO NANOMETRIC SCALE TOPOGRAPHY 429

TABLE 1
The Spread Area and Orientation of P388D1 Macrophages and Normal Rat Peritoneal Macrophages on Plain Fused Silica
Substrata and Gratings with Groove Depths of 30, 44, and 282 nm and Widths of 2 and 10 mm

Groove depth/ Mean area SD Orientation Orientation

width (mm) (mm2) (mm2) (mean angle) (variance)

P388D1 Plain 430 160 34 677

30/10 610 166 16 399
44/2 655* 151 10 212
44/10 663* 130 12 228
282/2 788** 193 6 61
282/10 741** 173 9 128
Peritoneal Plain 240 83 39 621
macrophages
(260) (100)
30/10 256 138 30 589
ConA (261) (129) (31) (601)
44/2 393 206 23 517
ConA (405) (230) (20) (308)
44/10 293 116 26 544
ConA (310) (137) (21) (348)
282/2 520* 181 19 212
ConA (532)* 204 (15) (156)
282/10 473* 206 21 354
ConA (510)* 196 (16) 238

Note. Rat peritoneal macrophages are given two values for cell area and orientation. The values in brackets are for macrophages which
were plated on substrata covered with ConA. Student’s t test was applied to compare the spread area of cells on plain substratum with the
area and orientation of cells spread on gratings.
* P õ 0.05; **P õ 0.01.

282 nm deep. Even substrata with very shallow
grooves, 30 nm deep, were able to induce rapid changes
in cell spreading and orientation. P388D1 macrophages
were firmly attached to the substratum and elongated
along groove/ridge boundaries.
The degree of orientation of cells increased with in-
creasing depth and with decreasing width of the
grooves. This was shown by the decreasing variance
and mean value of the orientation angle in cells grown
on patterned substrata (Table 1). The increase in cell
spread area in cells on patterned surfaces compared to
cells on smooth substrata was significant for all kinds
of gratings. However, although we observed a small
increase in cell spread area with increasing grove depth
and decreasing pitch, the effects of groove width and
depth on cell spread area were not statistically signifi-
cant.
The increase in cell spreading and orientation was
also observed in cells which encountered single steps
(Fig. 4a).
Normal peritoneal macrophages were also guided by
nanogrooves, although they were less sensitive to topo-
graphical guidance than P388D1 cells. A significant
increase in spread area was observed only in cells
plated on grooves deeper than 71 nm; shallower
grooves did not have a significant effect on the orienta-
tion and spread area of normal peritoneal macro-
phages. The effect of grooves on cell orientation is
shown in Table 1. Cell orientation increased with the
decreasing pitch and increasing depth of the gratings.
Since most of the nonactivated peritoneal macrophages
were very loosely attached to the gratings, we covered
the structure with concanavalin A. The treatment im-
proved cell adhesion and orientation on gratings (Ta-
ble 1).
F-actin Distribution and Content
F-actin in cells on plain substrata was concentrated
close to the cell margin and in a few areas where cells
extended pseudopodia (Fig. 2a). Cells were rounded
and not very well spread. Cells cultured on patterned
substrata showed a bright staining of F-actin concen-
trated along groove/ridge boundaries. The staining was
concentrated in places of cell attachment (Figs. 2b and
2c). TEM micrographs of transverse sections of P388D1
cells spread on grooves show an accumulation of elec-
tron-dense material localized in the cortical layer of
cytoplasm and related in its position to the groove/ridge
boundary (Figs. 5c and 5d).
The measurement of the F-actin content in P388D1
cells cultured for 15 min showed that cells on patterned
substrata contained two times more F-actin than cells
on plain substrata (Fig. 3). The F-actin content did

/ 6i0a$$3079 02-13-96 23:28:40 ecas AP: Exp Cell

430 WÓJCIAK-STOTHARD ET AL.

FIG. 2. F-actin distribution in P388D1 cells (a) on plain fused silica substrata; (b) P388D1 macrophage on gratings, 44-nm-deep grooves,
4-mm pitch; (c) normal peritoneal macrophage on gratings 71 nm deep; (d) confocal single plane section of macrophage on grooves 124 nm
deep, actin staining. Bars, 5 mm.

not depend on groove width but showed dependence on
groove depth.
Normal peritoneal macrophages showed the same
pattern of F-actin aggregation as P388D1 cells (Figs.
2c and 2d). We did not observe any regular arrange-
ment of F-actin in cells on grooves 30 and 40 nm deep
which correlated with the poor effect of these gratings
on cell orientation. Peritoneal macrophages cultured
on patterned substrata with a groove depth of 124 nm
and a width of 2 mm contained 18% SD 9% more F-
actin than cells cultured on plain substrata.
Vinculin, Membrane Receptors, and Phosphotyrosine
Staining
The staining of vinculin in P388D1 macrophages co-
localized with actin (Figs. 4b and 4c). Interference-re-
flection images of cells attached to the gratings showed
that actin and vinculin were found in places of close
apposition of the cell membrane to the substratum,
which corresponded to groove/ridge boundaries (Fig.
4d). This was mainly observed underneath the cell nu-
cleus and at the leading edge of the cell.
Phosphotyrosine staining in P388D1 macrophages
cultured on nanogrooves colocalized with actin and vin-
culin (Fig. 4f). Cells cultured on plain substrata showed
dot-like, punctuate staining of phosphotyrosine, which
did not correspond to the distribution of F-actin (Fig.
4e). Normal peritoneal macrophages which were
guided by the structure showed a similar pattern of
staining.
In preliminary studies we stained some of the cell

/ 6i0a$$3079 02-13-96 23:28:40 ecas AP: Exp Cell

 REACTION OF MACROPHAGES TO NANOMETRIC SCALE TOPOGRAPHY
FIG. 3. F-actin content (integrated brightness) in P388D1 macro-
phages cultured on plain and patterned substrata. Groove depth 44–
282 nm. *P õ 0.05.
membrane receptors which might be responsible for
binding cells to the substratum and subsequently bind-
ing actin and vinculin. We have not seen any regular
patterns of fibronectin receptor, a5b1 integrin, ICAM-
1, or a3b1 integrin in cells spread on gratings made in
plain fused silica or covered with ConA or fibronectin.
Vitronectin receptor, aVb1 integrin, was the only one
which showed a similar localization along groove/ridge
edges as actin and vinculin (Fig. 5).
Cell Adhesion
Gratings significantly increased the adhesion of
P388D1 cells, compared with planar surfaces. The ef-
fect was dependent on groove depth but not on width
(Fig. 6). More cells adhered to gratings with small
pitch. In normal peritoneal macrophages the effect of
gratings on cell adhesion was not significant.
Phagocytosis
P388D1 macrophages grown on patterned surfaces
phagocytosed more fluorescent microbeads than cells
grown on plain surfaces (Fig. 7). Contact of cells with
topography increased the number of active cells (cells
which showed phagocytosis) and increased the number
of phagocytosed beads/cell (Fig. 8). The effect depended
on groove depth but not on width.
DISCUSSION
Adhesive interactions between cells and the extracel-
lular environment play key roles in determining cell
/ 6i0a$$3079 02-13-96 23:28:40 ecas
431
morphology, gene expression, and the rate of cell prolif-
eration. It has been suggested by Gingell [14] that to-
pography itself can influence a spatial organization of
cytoskeleton-linked membrane receptors and, in turn,
affect several cell functions. Recently, we have found
that many kinds of cells are capable of sensing nanome-
tric topography and react to it by an increased adhe-
siveness and rearrangement of cell cytoskeleton (Curtis
et al., submitted for publication).
In this paper we observed substantial changes in the
morphology and behavior of macrophages during the
first 15 min of their attachment to patterned surfaces.
Cells acquired morphology typical of an activated mac-
rophage: they extended numerous microspikes and be-
came strongly attached to the substratum. Similar for-
mation of surface ruffles and microspikes was reported
for several types of cells in response to EGF, PDGF,
and also HGF/SF [15]. EGF, PDGF, and HGF/SF all
initiate a signal transduction cascade via the activation
of the receptor tyrosine phosphorylation [16]. We ob-
served an increase in the tyrosine phosphorylation in
macrophages spread on patterned substratum in places
colocalized with actin and vinculin. Tyrosine phosphor-
ylation was accompanied by an increase in the amount
of F-actin in cells spread along nanogrooves. Changes
in actin dynamics depend upon changes in actin-bind-
ing proteins (ABPs) associated with the microfilaments
of the leading edge [17]. Some of the ABPs, like ezrin
[17], spectrin [18], or annexin II [19], are phosphory-
lated on tyrosine in response to growth factors. These
proteins might also be potential mediators in cell reac-
tion to topography.
The way macrophages recognize ultrafine features
of topography remains unknown. We observed that in
macrophages spread on grooved substrata, F-actin, and
vinculin were localized along groove/ridge boundaries.
The interference-reflection image of the macrophage on
nanogrooved surface showed that places of close adhe-
sion are also localized along the groove edges. Electron
micrographs showed an extensive, electron-dense areas
localized close to the cell membrane at the groove/ridge
boundary. Since patterned fused silica substrata repre-
sent a chemically uniform surface, it is unlikely that
any components of the culture medium were selectively
adsorbed to the groove edges. This suggests that the
mechanical interaction of the groove edges with cell
membrane leads to selective adhesion which in turn
determines cell morphology and orientation. Since the
features of topography we used were as small as 30
nm, it is unlikely that the topography of the substra-
tum exerted a mechanical restriction on the locomotory
apparatus of the cell, which was proposed by Dunn and
Heath [20] as a mechanism of fibroblast guidance on
patterned substrata.
We report that the contact of macrophages with na-
AP: Exp Cell
432 WÓJCIAK-STOTHARD ET AL.

/ 6i0a$$3079 02-13-96 23:28:40 ecas AP: Exp Cell

 REACTION OF MACROPHAGES TO NANOMETRIC SCALE TOPOGRAPHY 433

FIG. 5. Distribution of vitronectin receptors, aV integrins, in P388D1 macrophages cultured on (a) plain substratum and (b) patterned
substratum, groove width 2 mm. Bar, 5 mm. (c, d) Electron micrographs of a P388D1 macrophages spread over a step 140 nm high. Transverse
sections. (c) A 150-nm-thick section and (d) a 60-nm-thick section; g, groove; r, ridge; the arrow points to the electron-dense areas at the
groove/ridge boundary. Bars, 100 nm.

nogrooves increases their phagocytotic activity. In mac-
rophages, activation of C3 receptors occurs via inte-
grin–ligand interactions [14]. It was shown by Wright
et al. [21] and Bohnsack et al. [22] that the adhesion of
macrophages to surfaces coated with covalently bound
fibronectin or laminin increases their phagocytotic ac-
tivity. We report an accumulation of aV integrins, colo-
calized with vinculin and F-actin along groove/ridge
boundaries in cells spread on patterned substrata. This
suggests that the activation of macrophages by topog-

FIG. 4. Vinculin and tyrosine kinase distribution in cells grown on patterned substrata. (a) Alignment of P388D1 cells along a single
step, height 44 nm. Bar, 20 mm. (b) Vinculin in macrophage reacting with a single step 44 nm high. Bar, 5 mm. (c) Vinculin staining in
P388D1 macrophage on gratings 71 nm deep. Bar, 5 mm. (d) Interference-reflection image of a macrophage on gratings, 71 nm deep. Darker
places correspond to the cell membrane being closer to the substratum. The image was obtained with the use of 100X objective, recorded
by the low light level camera and photographed from the computer monitor. Bar, 5 mm. (e) Distribution of proteins with phosphotyrosine
residues in a macrophage on plain substratum and (f) in a macrophage on gratings 71 nm deep. Cells were fixed and stained 15 min after
plating. Bars, 5 mm.

/ 6i0a$$3079 02-13-96 23:28:40 ecas AP: Exp Cell

434 WÓJCIAK-STOTHARD ET AL.
FIG. 6. Cell adhesion to plain and grooved substrata. The num-
ber of cells/field was counted for each individual type of gratings 15
min after cell plating. *P õ 0.05.
raphy may be a consequence of clustering of specific
membrane receptors. Clustering of b1 integrins has
been shown to induce tyrosine phosphorylation in sev-
eral kinds of cells [23, 24]. This finding is consistent
with our observations of an increased tyrosine phos-
phorylation in macrophages spread on nanogrooves.
FIG. 7. Phagocytosis of P388D1 cells cultured on plain and pat-
terned surfaces. Cells were cultured for 5 h with a suspension of
fluorescent beads. The number of beads phagocytosed by 100 cells
on plain and patterned surfaces was counted. Student’s t test was
used to compare phagocytosis of cells on plain and patterned sur-
faces. *P õ 0.05.
/ 6i0a$$3079 02-13-96 23:28:40 ecas
FIG. 8. Percentage of P388D1 cells which phagocytosed certain
number of beads (0, 1, and more than 2) in cell populations grown
on plain surface and cell populations grown on grooves 2 mm wide,
71 nm, and 282 nm deep.
In conclusion, we found that murine macrophages
show very high sensitivity to surface topography and
are guided and activated by features as small as 30–
70 nm. Since this size corresponds to the size of a single
collagen fiber, macrophages are likely to show a similar
type of reaction to the topography of the extracellular
matrix in vivo. Contact of cells with topographical fea-
tures induces rapid changes in cell shape, increases the
content of F-actin in cells, and increases their phagocy-
totic activity and adhesiveness. The reaction is likely
to be induced by a selective clustering of integrins on
topographical features followed by the phosphorylation
of tyrosine. The reaction of cells to substratum surface
topography shows several similarities with the re-
sponse of cells to growth factors.
The authors’ work was supported by a SERC grant and by a grant
from the Wellcome Trust for a laser scanning microscope. Dr. Beata
Wojciak-Stothard is a Research Assistant on leave from the Depart-
ment of Cell Biology, Institute of Molecular Biology, Jagiellonian
University, Cracow. The authors thank Dr. L. Tetley and Ms. M.
Mullin (Glasgow University) for their very kind help and expertise
with the transmission electron microscope as well as Ms. M. Robert-
son and Mr. J. McGadey (Glasgow University) for their kind assis-
tance and help with the use of the scanning electron microscope.
REFERENCES
1. Dunn, G. A., and Ebendal, T. (1978) Exp. Cell Res. 101, 1–14.
2. Haston, W. S., Shields, J. M., and Wilkinson, P. C. (1983) Exp.
Cell Res. 146, 177–126.
3. Wilkinson, P. C., and Lackie, J. M. (1983) Exp. Cell Res. 145,
255–264.
AP: Exp Cell
 REACTION OF MACROPHAGES TO NANOMETRIC SCALE TOPOGRAPHY
4. Gallin, J. I., and Quie, P. (1978) Leukocyte Chemotaxis: Meth-
ods, Physiology, and Clinical Implications. Raven Press, New
York.
5. Clark, P., Connolly, P., Curtis, A. S. G., Dow, J. A. T., and
Wilkinson, C. D. W. (1990) Development 108, 635–644.
6. Curtis, A. S. G., and Clark, P. (1990) Crit. Rev. Biocompatibility
5, 343–362.
7. Clark, P., Connoly, P., Curtis, A. S. G., Dow, J. A. T., and Wilkin-
son, C. D. W. (1991) J. Cell Sci. 99, 73–77.
8. Brunette, D. M. (1986) Exp. Cell Res. 164, 11–26.
9. Wójciak, B., Crossan, J., Curtis, A. S. G., and Wilkinson,
C. D. W. (1995) J. Mater. Sci. 6, 266–271.
10. Wójciak-Stothard, B., Madeja, Z., Korohoda, W., Curtis, A., and
Wilkinson, C. (1995) Cell Biol. Int. Rep., in press.
11. Curtis, A. S. G., Wilkinson, C., and Wojciak-Stothard, B. (1995)
Cell. Eng. Inc. Mol. Eng., in press.
12. Dunn, G. A., and Brown, A. F. (1986) J. Cell Sci. 83, 313–340.
13. Van Furth, R., and Diesselhoff-Den Dulk M. M. (1980) Scand.
J. Immunol. 12, 265–269.
14. Gingell, D. (1993) Contact Signalling and Cell Motility (Jones,
G., Wigley, C., and Warn, R., Eds.), pp. 1–33, SEB Symposium
No. 47.
Received August 7, 1995
Revised version received November 27, 1995
/ 6i0a$$3079 02-13-96 23:28:40 ecas
435
15. Warn, R., Brown, D., Dowrick, P., Prescott, A., and Warn, A.
(1993) in Cell Behaviour: Adhesion and Motility (Jones, G., Wig-
ley, C., and Warn, R., Eds.), pp. 325–338, SEB Symposium No.
47.
16. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B.,
Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281–
302.
17. Gould, K. L., Cooper, J. A., Bretsher, A., and Hunter, T. (1986)
J. Cell Biol. 102, 660–669.
18. Bretscher, A. (1989) J. Cell Biol. 108, 921–930.
19. Hunter, T., and Cooper, J. A. (1981) Cell 24, 741–752.
20. Dunn, G. A., and Heath, J. P. (1976) Exp. Cell Res. 101, 1–14.
21. Wright, S. C., Craigmyle, L. S., and Silverstein, S. C. (1983) J.
Exp. Med. 158, 1338–1343.
22. Bohnsack, J. F., Kleinmann, H. K., Takahashi, T., O’Shea, J. J.,
and Brown, E. J. (1985) J. Exp. Med. 161, 912–923.
23. Kornberg, L. J., Earp, H. S., Turner, C. E., Prockop, C., and
Juliano, R. L. (1991) Proc. Natl. Acad. Sci. USA 88, 8392–8396.
24. Hynes, R. O. (1992) Cell 69, 11–25.
25. Chou, L., Firth, J. D., Uitto, V. J., and Brunette, D. M. (1995)
J. Cell Sci. 108, 1563–1573.
AP: Exp Cell