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J. Cell Set. 50,
1-7
(1981)
Printed in Great Britain
©
Company of
Biologists
Limited ig8i
ANOMALOUS PREFERENCES OF CULTURED
MACROPHAGES FOR HYDROPHOB1C AND
ROUGHENED SUBSTRATA
ABBY RICH*
AND
ALBERT K. HARRIS
Department
of
Zoology,
University
of North
Carolina,
Chapel
Hill,
N.C.
27514, U.S.A.
SUMMARY
Peritoneal macrophages were plated
out on a
series
of
artificial haptotactic substrata con-
sisting
of
grid patterns
of
vacuum-evaporated palladium metal alternating with hydrophobic
untreated polystyrene and with hydrophilic sulphonated polystyrene. By their active locomo-
tion
the
macrophages accumulated preferentially onto
the
less hydrophilic
of
either pair
of
these alternative substrata. This order
of
substratum preference
is
precisely
the
opposite
to
that shown
by
fibroblastic cells. Macrophages were also found
to
accumulate preferentially
onto roughened culture surfaces as opposed
to
smooth ones, which
is
again opposite
to the
behaviour
of
fibroblasts. These opposite substratum preferences shown by macrophages may
reflect physical
as
well
as
functional differences
in
their surfaces,
and
could serve
as
assay
criteria for macrophages and cell types putatively equivalent to macrophages.
INTRODUCTION
Tissue-cell locqmotion requires a substratum and the properties of this substratum
can strongly influence the locomotion that occurs.
For
example, Carter (1967a, b)
showed that fibroblasts migrate preferentially from cellulose acetate
to
thin vacuum-
evaporated layers of palladium metal. In a later study (Harris, 1973), both normal and
transformed fibroblasts were found to migrate onto sulphonated polystyrene (as well
as onto glass in preference to palladium, but onto palladium in preference to untreated
polystyrene).
In each
of
these cases the cells showed
a
consistent hierarchy
of
relative adhesive
preference, accumulating
on
the more hydrophilic
of
any pair
of
substances. More
recently, Letourneau (1975) showed that nerve growth cones obey this same hierarchy
of substratum preference and can be guided along paths of greater adhesiveness. This
demonstration lends support
to
theories
of
nerve guidance
by
adhesive differences
(Barbera, Marchase & Roth, 1973).
In those previous studies, each cell type showed the same sequence of relative pre-
ference
for
substrata. We have now studied the adhesive preferences
of
the macro-
phage,
a
cell type whose behaviour was already known
to be
unusual
in
several
respects. Macrophages migrate invasively
as
part
of
their normal function, fail
to
show contact inhibition
of
locomotion (Oldfield, 1963),
and
even show
a
sort
of
Present
address:
Department of
Cell
Biology, New York University Medical Center, New
York, N.Y. 10016, U.S.A.
2 A Rich and A. K. Harris
reverse contact inhibition relative to fibroblasts (Harris, 1974). We have found that
macrophages also show significant differences in their adhesive preferences.
MATERIALS
AND
METHODS
Peritoneal macrophages were obtained from Swiss white mice, which were killed
by
cervical
dislocation
and the
cells collected
in 3-4
ml
of
phosphate buffered saline (PBS) (Dulbecco
&
Vogt, 1954).
In
some experiments, heparin was included
in the
saline,
but
seemed
to
make
no
difference
to the
results. After centrifugation,
the
cells were resuspended
in
PBS
and
allowed
to settle onto
the
experimental substrata
for
15-20 min
at
37 °C, before replacing
the
saline
with Dulbecco's modified Eagles medium buffered with 25 mM-.N-2-hydroxyethylpiperazine-
N'-ethanesulfonic acid (HEPES),
and
supplemented with
10%
foetal calf serum (GIBCO,
Grand Island, N.Y.)
and
penicillin
and
streptomycin.
For
certain experiments, 5
ml of
thio-
glycollate medium (Fisher Scientific Co.) was injected intraperitoneally 5 days prior
to
collect-
ing macrophages
as
above.
A
culture
of
the macrophage-like cell line, P338D,, was
a
gift from
Dr H.
S.
Koren, Duke University. WI38 cells, VA13 cells (a line
of
untransformed fibroblasts),
simian virus-transformed VA13 cells,
and
L929 cells
(a
transformed line
of
mouse fibroblasts)
were
all
obtained from
the
Tissue Culture Laboratory, Cancer Research Center, University
of
North Carolina
at
Chapel Hill.
H615 ts 3T3
cells were
a
generous gift from
Dr
Kenneth
Noonan
of
the University
of
Florida.
Three artificial substrata were used:
(1)
untreated polystyrene dishes, 35
mm in
diameter;
(2) polystyrene dishes treated with concentrated sulphuric acid
for
2
h to
make
the
substratum
hydrophilic (Maroudas, 1975);
and (3)
palladium evaporated onto treated
and
untreated poly-
styrene (Carter, 1967ft).
All 3 of
these culture surfaces could
be
produced side-by-side
in
individual Petri dishes. Approximately 2
x io
5
macrophages were plated
on
1 dish.
Slightly roughened regions were created
on
Falconized polystyrene surfaces
of
tissue culture
flasks by gently sliding
the
round, fire-polished
end of
a
glass
rod
across
the
surface so that
the
rough areas were just barely visible
by
phase-contract microscopy. Approximately 2
x io
6
cells
were plated
per
25 cm* flask.
Cultures were viewed with
an
Olympus
CK
inverted phase-contrast microscope
and
photo-
graphed with
an
Olympus 35
mm
camera using Kodak Panatomic
X
film. Individual frames
from
a
16 mm Bolex camera using Plus
X
reversal film were converted
to
120 film
by
a Testrite
cine-enlarger. During observations,
a
Sage air-current incubator maintained
the
temperature
of the cultures
at
37
C
C.
RESULTS
When plated out on grid patterns of alternative substrata, both the transformed
(VA13 and L929) and untransformed (WI38 and ts 3T3) fibroblasts migrated pre-
ferentially onto the more hydrophilic material available to them (i.e. untreated poly-
styrene < palladium < sulphonated polystyrene). Peritoneal macrophages, however,
developed the opposite sequence of relative preference to these 3 substrata.
Macrophages accumulated on untreated polystyrene in preference to palladium
and on palladium in preference to sulphonated polystyrene (Fig.
1
c,
D).
In addition,
a greater percentage of macrophages flattened themselves onto the more preferred
substrata. This reversed pattern of accumulation occurred when foetal calf serum or
beef heart infusion was used to supplement the medium.
Another unusual characteristic of macrophages is the length of time required for
these reversed-substratum preferences to develop. Fibroblasts accumulated on their
preferred substrata within 1 day but macrophages required between 1 and 2 days
Macrophage substratum preferences %
to accumulate onto palladium in preference to sulphonated polystyrene, and 7 days
to develop their preference for untreated polystyrene over palladium. This delay
could not be explained by an insufficient amount of cell locomotion. Time-lapse films
showed that the macrophages moved extensively during this 7-day delay.
To test whether this delay might be caused by a gradual deposition of some secre-
tory product, macrophages were cultured for 2 weeks on one of the untreated poly-
styrene-palladium substrata and then removed with 4% EDTA. Fresh macrophages
•1c
Fig. 1.
A.
Accumulation of ts 3T3 fibroblasts on palladium (dark squares) in prefer-
ence to untreated polystyrene; and
B,
on sulphonated polystyrene in preference to pal-
ladium (dark squares), c, Accumulation of macrophages after 7 days in culture on
untreated polystyrene in preference to palladium (dark squares); and
D,
on palladium
(dark squares) in preference to sulphonated polystyrene. Bar, o-i mm.
were then plated onto the previously occupied substratum, and they also required
7 days to develop their preference for the more hydrophobic surface. This result shows
that whatever change occurs is intrinsic to the cells. In addition, VA13 fibroblasts
were plated out on the same substrata with macrophages and still showed the same
sequence of relative preference.
We also considered whether the change in macrophage substratum preference
might be related to the set of reactions they can undergo, known as activation. To
test the possibility, macrophages were pre-stimulated by an intraperitoneal injection
of thioglycollate medium. These macrophages did spread faster than normal ones but
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