Published May 1, 1983
Production of Laminin and Fibronectin by
Schwannoma Cells: Ceil-Protein Interactions In Vitro
and Protein Localization in Peripheral Nerve In Vivo
SALLY I_. PALM and LEO T. FURCHT
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis,
Minnesota 55455
Schwann cells are the supporting structures for nerve processes
in the peripheral nervous system. In myelinated nerves, the
Schwann cells form the myelin sheath. Schwann cells are
associated with a basement membrane. From in vivo and in
vitro studies of neurons and Schwann cells, it has been found
that basement membrane is produced by the Schwann cells (13). The nerve sheath formed by Schwann cells is important in
proper regeneration of damaged axons (4). In vitro, Schwann
ceils do not produce a basement membrane until they have
become committed to an axon or another Schwann cell (5).
Nerve cells, which do not produce a basal lamina in culture,
appear to exert some trophic influence on Schwann cells,
stimulating the Schwann cells to produce basement membrane
and collagen fibrils (3).
1218
Basement membranes, such as produced by Schwann cells,
serve several functions in the body. Basement membranes form
the semipermeable membrane of the kidney glomerulus and
act as supporting structures in the lens capsule of the eye and
under vascular endothelium. Basement membranes can serve
as a matrix or form to guide tissue regeneration (for review, see
reference 6). At present, there are three known major constituents of basement membrane: collagens, specifically type IV
collagen, possible some type V collagen; noncollagenous glycoproteins, among which are laminin, fibronectin, and entactin
(7-10); and glycosaminoglycans, such as heparan sulfate (I 1).
Laminin was first isolated from the mouse EHS tumor (12),
and independently from a mouse embryonal carcinoma-derived cell line, where it was called GP-2 (13). Laminin is a high
THE JOURNAL Of CELL BIOLOGY • VOLUME 96 MAY 1983 1218-1226
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ABSTRACT We studied a rat Schwannoma cell line (RN22F) to determine if it produced the
basement membrane glycoproteins laminin and fibronectin, and how it interacted with these
proteins in vitro. We used antisera to laminin and fibronectin for immunoprecipitation
experiments and immunncytochemical localization at the electron microscope level. Polyacrylamide gels of antilaminin immunoprecipitates of conditioned medium and solubilized Schwannoma cells contained bands of reduced Mr 200,000 and 150,000. Antilaminin immunoprecipitates of conditioned medium contained nonreduced bands of 850,000 daltons and 150,000, and
immunoprecipitates of solubilized cells contained nonreduced bands of 850,000, 400,000,
200,000, and 150,000 daltons. Antifibronectin immunoprecipitates of conditioned medium
contained a reduced band of 220,000 daltons, and nonreduced bands of 440,000 and 220,000
daltons. Radio-labeled protein was not detected in antifibronectin immunoprecipitates of
solubilized cells. By immunocytochemistry, laminin was found along the cell surface in a
continuous band, whereas fibronectin was only sparsely distributed along the cell surface. In
cell adhesion assays, Schwannoma cells bound preferentially to laminin-coated substrates as
compared to fibronectin or noncoated substrates. A number of Schwannoma cells displayed a
curved and elongated morphology on laminin substrates, as compared to a uniformly spread
morphology on fibronectin, and a round, nonspread morphology on noncoated substrates.
Immunofluorescent staining showed laminin in the endoneurium and perineurium and fibronectin predominantly in the perineurium of mouse sciatic nerve in vivo. The production of
laminin and fibronectin by Schwann cells may be important in the development and myelination of peripheral nerves, and the proper regeneration of axons following nerve injury.
Published May 1, 1983
MATERIALS AND
METHODS
Cells: Cells used in this study were line RN22F, a clonal line of rat
Schwannoma ceils. The cells were a gift from Dr. John R. Sheppard (Dight
Institute of Genetics, University of Minnesota). This clone was derived in Dr.
Sheppard's lab from line RN22, a rat Schwannoma cell line originally established
from Schwann tumors (neurilemmomas) of rats induced chemically by Dr. S. E.
Pfeiffer (University of Connecticut). The RN22F dnnal line retains the ability of
the parental RN22 line to respond to cyclic nucleotide analogs. The RN22F line
also produces material which, when bound to substrate, can act as a neuritepromoting factor for neurons of dissociated chick dorsal root ganglia (S. Rogers,
P. Letourneau, and S. Palm, unpublished observations). The RN22F cells were
maintained in Dulbecco's Modified Eagle's Medium (DME) (Gibeo Laboratories,
Grand Island Biological Co., Grand Island, NY), with 10% heat inactivated horse
serum (Sterile Systems, Inc., Logan, UT).
Protein Isolation: Fibronectin was isolated from human plasma using
gelatin affinity chromatography, as previously described (36). Laminin was
isolated from the mouse EHS tumor (kindly provided by Dr. George Martin
[National Institutes of Health]) using a modification of the method of Timpl et
al. (12). Tumor was homogenized in 3.4 M NaC1, 0.01 M phosphate buffer pH
7.4, with 50 #g/ml phenylmethylsulfonylfluoride (PMSF) (Sigma Chemical Co.,
St. Louis, MO) and 50/.tg/ml p-hydroxymercuribenzoate (Sigma Chemical Co.),
washed once with the same buffer, then extracted overnight with 0.5 M NaC1,
0.01 M phosphate, pH 7.4, and 50 #g/ml each of PMSF and p-hydroxymercuribenzoate. Type IV collagen was removed by raising the salt concentration to 1.7
M followed by stirring for 1 h at 4°C, and centrifugation (10,000 rpm, 30 rain).
Laminin was precipitated from the extract with 30% saturation ammonium
sulfate. The precipitate was resuspended in 0.5 M NaC1, 0.01 M phosphate, pH
7.4, and dialyzed against the same buffer. Laminin was isolated from the
precipitate by gel filtration chromatography on Sephacryl S-300 (Pharmacia Fine
Chemicals, Piscataway, NJ) (2.6 ::< 1130cm column) in the 0.5 M NaC1 buffer,
where it eluted just after the void volume. The laminin containing peak was
dialyzed against 0.14 M NaC1, 0.01 M phosphate buffer, pH 7.4 (phosphatebuffered saline [PBS]), then chromatographed on a heparin-Sepharose aff'mity
column (Pharmacia Fine Chemicals) equilibrated with PBS, and eluted with 0.5
M NaCI, 0.01 M phosphate, pH 7.4. The protein solution was concentrated by
evaporation while in a dialysis bag, redialyzed against 0.5 M NaCI, 0.01 M
phosphate, pH 7.4, and stored at -70°C or filter sterilized with a Millex 0.45-~m
pore sterile filter (Millipore Corp., Bedford, MA) and refrigerated.
Antibody Production and Isolation: We immunized New Zealand White rabbits with multiple subcutaneous injections of laminin and fibronectin. Antilaminin antiserum showed no reactivity against type IV collagen or
fibronectin by enzyme-hnked immunosorbent assay (ELISA). Antifibronectin
antiserum was affinity purified on fibronectin agarose. Affinity purified antilaminin antiserum (a gift from Dr. Michael Silver, University of Minnesota) was
purified over laminin, fibronectin, and type IV collagen affinity columns. Antiserum to entactin was a generous gift from Dr. Albert Chung (University of
Pittsburgh).
Immunoprecipitation: Confluent RN22F cultures in 35-ram tissue
culture plates (Falcon 3001; Falcon Labware, Oxnard, CA) were radio-labeled
by overnight incubation with 1 ml of serum free medium containing 50/sCi/ml
[SH]leucine (110 Ci/mmol) or :~H-aminoacids (both from New England Nuclear,
Boston, MA). Conditioned medium was collected, the cell layer washed with PBS
with Ca ++ and Mg+÷, and the cells solubilized with 1 ml per plate of 100 mM
NaCI, 2 mM EDTA, 10 mM Tris, pH 7.2, 1% Nonidet P-40 (NP-40, Particle
Data Services, Inc., Elmhurst, IL), 0.5% sodium deoxycholate and I% Trasylol
(Sigma Chemical Co.). The cell extract was clarified by centrifugation for 1 h at
15,000 rpm. For each sample, ceils or medium from two 35-mm plates were
pooled, and the solutions incubated with 20 #1 of antiserum or pre-immune
control serum for 30 min at 22°C. Immune complexes were adsorbed by adding
200 #1of a 10% suspension of washed Protein A-containing Staphylococcusaureus
(Pansorbin, Calbiochem-Behring Corp., La Jolla, CA). The bacteria/immune
complexes were washed once in I M NaCI, 10 mM Tris, pH 7.2, 1% NP-40, and
twice in 150 mM NaCl, 10 mM Tris, pH 7.2, 1% Triton X-100 and 1% SDS.
Precipitates were dissociated under reducing or nonreducing conditions. For
reducing conditions, samples were heated for 1 min, 100°C. in sample buffer
containing 0.08 M Tris, pH 6.8, 3% SDS, 15% glycerol, 5% mercaptoethanol, and
0.01% bromophenol blue. For nonreducing conditions, samples were mixed at
22°C with buffer containing 1 M glacial acetic acid, 3% SDS. 15% glycerol, and
0.01% bromophenol blue. The bacteria were removed by centrifugation and
supernatants apphed to polyacrylamide gels consisting of a 2% stacking gel on a
2-10% gradient running gel. Gels were prepared using the buffer system of
Laemmli (37) and acrylamide formulations of Blattler et al. (38). Gels were
stained with Coomassie Blue G-250 (Kodak) treated with En:3Hance (New
England Nuclear, Boston, MA) and dried. Fluorography was performed using
Kodak XAR5 autoradiography film.
Immunocytochemistry on RN22F Cells: RN22F cells were
grown to confluence and processed for immunocytochemistry as described elsewhere (39). Antisera to laminin and fibronectin, and the pre-immune rabbit
serum control were used at 1:100 dilution in PBS with 1% goat serum. Secondary
antiserum (goat anti-rabbit IgG, Cappel Laboratories, Cochranville, PA) was
used at 1:250 dilution, and rabbit peroxidase anti-peroxidase (Cappel Laboratories) at I:1,000 dilution. Diaminobenzidine reaction, postfixation, and Epon
embedment were performed as described (39). Sections were viewed on a Philips
EM 300 transmission electron microscope.
Cell-binding Assay: Plastic petri dishes (Falcon 1008, 35 mm) were
coated with protein by incubatijng with I ml of protein-containing solution at a
given concentration in 0.05 M sodium carbonate buffer, pH 9.6, for 3 h at 37°C.
Plates were prepared fresh for each assay. To quantitate the amount of protein
that actually bound to the plates, a set of plates was made using laminin and
fibronectin labeled with 3H using the reductive methylation technique of Jentoft
and Dearborn (40). Nonbound protein was removed by washing with PBS, and
bound protein solubilized in 0.5 N NaOH, 1% SDS, added to Aquasol I1 (New
England Nuclear) and counted in a Beckman LS230 scintillation counter (Beckman Instruments, Inc., Palo Alto, CA). The counts bound to the plates were
compared with total counts available in the protein solution in order to estimate
the amount of protein bound.
Cells for the assay were trypsinized from culture flasks, washed once in DME
with 10% horse serum, and twice in serum free DME. Ceils were counted and
adjusted to 1 x 10~ ceUs/ml in serum free DME, added to each plate in a volume
of 1 ml and incubated without agitation for 90 rain in a 37°C humidified
incubator with 5% CO2, 95% air atmosphere. Nonbound ceUs were removed from
the plates by washing x 5 with PBS with Ca ++ and Mg++. Bound cells were
quantitated by one of two methods. Noaradio-labeled cells were trypsinized from
the assay plates, and the number of ceils counted with a Coulter counter.
PALMAND FURCHT Laminin and Fibronectin in Schwannoma Cells
1219
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m o l e c u l a r w e i g h t g l y c o p r o t e i n o f M~ 850,000 o r g r e a t e r (14,
15). L a m i n i n is c o m p o s e d o f d i s u l f i d e - b o n d e d s u b u n i t c h a i n s
o f 4 0 0 a n d 200 k d a l t o n s , w i t h a p o s s i b l e s t o i c h i o m e t r y o f o n e
4 0 0 - k d a l t o n c h a i n to t h r e e 2 0 0 - k d a l t o n c h a i n s (12, 15). I n vivo,
l a m i n i n h a s b e e n f o u n d in b a s e m e n t m e m b r a n e s t h r o u g h o u t
the body, including the glomerular basement membrane, vascular endothelial basement membrane, and underlying the
e p i t h e l i u m o f t h e s k i n (7, 8). L a m i n i n p r o m o t e s t h e a t t a c h m e n t
o f e p i t h e l i a l cells to t y p e IV c o l l a g e n (16), a n d i n c r e a s e s t h e
a d h e s i o n o f c a r c i n o m a cells to s u b s t r a t e s in vitro (17). C e l l s
f r o m r e g e n e r a t i n g liver s h o w e n h a n c e d a d h e s i o n to l a m i n i n
d u r i n g t h e r e g e n e r a t i o n p e r i o d (18). A n u m b e r o f cell lines
g r o w n in vitro p r o d u c e l a m i n i n , i n c l u d i n g cells f r o m e n d o t h e lium, embryonal carcinomas, parietal yolk sac tumor, breast
a d e n o c a r c i n o m a , rat liver e p i t h e l i u m , a n d rat b r e a s t e p i t h e l i u m . T h e l a m i n i n m o l e c u l e c a n b i n d to g l y c o s a m i n o g l y c a n s
(19), p a r t i c u l a r l y h e p a r i n (20).
F i b r o n e c t i n is a h i g h m o l e c u l a r w e i g h t g l y c o p r o t e i n o f M r
440,000 with two apparently similar disulfide-bonded chains
o f ~ 2 2 0 , 0 0 0 . F i b r o n e c t i n is f o u n d in p l a s m a , a n d is a m a j o r
c o m p o n e n t o f b a s a l l a m i n a , c o n n e c t i v e tissue, a n d t h e e x t r a c e l l u l a r m a t r i x p r o d u c e d b y f i b r o b l a s t s in vitro a n d in v i v o (for
r e v i e w s , s e e r e f e r e n c e s 2 1 - 2 4 ) . F i b r o n e c t i n is i n v o l v e d in t h e
a d h e s i o n o f v a r i o u s cells to c o l l a g e n in vitro, i n c l u d i n g fibrob l a s t s , C h i n e s e h a m s t e r o v a r y cells, a n d p l a t e l e t s ( 2 5 - 2 7 ) . T h e
f i b r o n e c t i n m o l e c u l e b i n d s to f i b r i n (28), h e p a r i n (29), a n d
s t a p h y l o c o c c i (30). I n d e v e l o p m e n t , f i b r o n e c t i n m a y p l a y a role
i n m u s c l e m o r p h o g e n e s i s (31), in t h e i n t e r a c t i o n o f m e s e n c h y m a l cells w i t h c o l l a g e n o u s m a t r i c e s d u r i n g e n d o c h o n d r a l b o n e
f o r m a t i o n (32), a n d i n t h e s p a t i a l o r g a n i z a t i o n o f cells in
d e v e l o p i n g c h i c k w i n g (33).
A c l o n a l line o f rat S c h w a n n cells ( R N 2 ) h a s b e e n d e s c r i b e d
t h a t s y n t h e s i z e s c o l l a g e n s in vitro (34). A s u b c l o n e o f this line
( R N 2 2 ) u n d e r g o e s a n a p p a r e n t d i f f e r e n t i a t i o n , as e v i d e n c e d b y
m o r p h o l o g i c a l c h a n g e , w h e n t r e a t e d w i t h cyclic n u c l e o t i d e
a n a l o g u e s (35). I n this s t u d y , w e e x a m i n e d cell line R N 2 2 F to
d e t e r m i n e i f t h e line m a d e n o n c o l l a g e n o u s b a s e m e n t m e m b r a n e a s s o c i a t e d p r o t e i n s , a n d w h e t h e r t h e s e cells i n t e r a c t e d
w i t h b a s e m e n t m e m b r a n e p r o t e i n s in vitro.
Published May 1, 1983
RESULIS
lmmunoprecipitation
R E D U C E D GELS: I m m u n o p r e c i p i t a t e s o f R N 2 2 F c o n d i tioned medium using antilaminin antiserum produced major
b a n d s at 200 a n d 150 k d a l t o n s w h e n c o m p l e x e s w e r e dissoc i a t e d u n d e r r e d u c i n g c o n d i t i o n s (Fig. 1 b, l a n e 3). T h e 200-
FIGURe I 2-10% SDS polyacrylamide gradient gel. (a) Purified EHS
laminin. R, reduced. NR, nonreduced. Coomassie Blue stain. (b)
Fluorograph of immunoprecipitates of conditioned medium from
RN22F cultures metabolically labeled with [aH]leucine. Reducing
sample buffer. Lane I, pre-immuneserum. Lane 2, antifibronectin
antiserum. Lane 3, antilaminin antiserum. (c) Fluorograph of immunoprecipitates of solubilized RN22F cell layer metabolically labeled with [aH]leucine. Reducing sample buffer. Lanes are as in b.
1220
THE JOURNAL OF CELL BIOLOGY. VOLUME 96, 1983
FIGURE 2 2-10% SDS polyacrylamide gradient gel. (a) Purified EHS
laminin. R, reduced. NR, nonreduced. Coomassie Blue stain. (b)
Fluorograph of immunoprecipitates of conditioned medium from
RN22F cultures metabolically labeled with [aH]leucine. 1 M acetic
acid sample buffer. Lane 1, pre-immune rabbit serum. Lane 2,
antifibronectin antiserum. Lane 3, antilaminin antiserum. (c) Fluorograph of immunoprecipitates of solubilized RN22F cell layer metabolically labeled with [DH]leucine. 1 M acetic acid sample buffer.
Lanes are as in b.
k d a l t o n b a n d c o - m i g r a t e d w i t h t h e 2 0 0 - k d a l t o n b a n d o f red u c e d p u r i f i e d l a m i n i n f r o m t h e E H S t u m o r (Fig. 1 a, lane R).
A n t i f i b r o n e c t i n a n t i s e r u m p r e c i p i t a t e d p r o t e i n o f 220 k d a l t o n s
f r o m c o n d i t i o n e d m e d i u m (Fig. 1 b, lane 2). T h i s b a n d comigrated with reduced human plasma fibronectin.
A n t i l a m i n i n p r e c i p i t a t e s o f t h e solubilized cell layer p r o d u c e d a m a j o r b a n d at 200 k d a l t o n s a n d a w e a k e r b a n d at 150
k d a l t o n s (Fig. 1 c, lane 3). A n t i f i b r o n e c t i n a n t i s e r u m d i d n o t
p r e c i p i t a t e a d e t e c t a b l e a m o u n t o f p r o t e i n f r o m the solubilized
cells (Fig. 1 c, lane 2). N o r m a l r a b b i t s e r u m d i d n o t p r e c i p i t a t e
a n y p r o t e i n f r o m c o n d i t i o n e d m e d i u m o r solubilized cells (Fig.
1 b a n d c, lanes 1). A faint e x t r a b a n d at 220 k d a l t o n seen in
all t h r e e lanes o f Fig. 1 b is d u e to s m a l l a m o u n t s o f f i b r o n e c t i n
b i n d i n g directly to t h e Staphylococcus w i t h o u t the a n t i b o d y
i n t e r m e d i a t e , as h a s b e e n s h o w n b y o t h e r g r o u p s p r e v i o u s l y
(30, 43). A n t i e n t a c t i n a n t i s e r u m did n o t p r e c i p i t a t e d e t e c t a b l e
a m o u n t s o f entactin, a 158-kdalton protein, or t h e 150-kdalton
b a n d seen u s i n g a n t i l a m i n i n a n t i s e r u m , f r o m solubilized cells
or medium.
N O N R E D U C E D GELS: A 1 M acetic acid s a m p l e b u f f e r
w a s u s e d to d i s s o c i a t e c o m p l e x e s w i t h o u t r e d u c i n g disulfide
b o n d s . U s i n g this s a m p l e buffer, w e f o u n d t h a t a n t i l a m i n i n
immunoprecipitates of RN22F-conditioned medium contained
b a n d s o f 850 a n d 150 k d a l t o n s (Fig. 2 b, lane 3). T h e 850kdalton band co-migrated with nonreduced purified laminin
f r o m t h e E H S t u m o r (Fig. 2 a, l a n e NR). A n t i f i b r o n e c t i n
i m m u n o p r e c i p i t a t e s o f m e d i u m c o n t a i n e d b a n d s o f 440 a n d
220 k d a l t o n (Fig. 2 b, lane 2) c o n s i s t e n t w i t h t h e d i m e r i c a n d
m o n o m e r i c f o r m s o f f i b r o n e c t i n , respectively. T h e 4 4 0 - k d a l t o n
b a n d c o - m i g r a t e d w i t h h u m a n p l a s m a f i b r o n e c t i n r u n in acetic
acid s a m p l e b u f f e r ( d a t a n o t s h o w n ) . A n t i l a m i n i n i m m u n o p r e cipitates o f t h e s o l u b i l i z e d cell l a y e r gave a m a j o r b a n d at 850
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Alternatively, cells were radio-labeled with [aH]thymidine (5 /tCi/ml, 6.7 Ci/
mmol) for 48 h, or 3H-amino acids (25 #Ci/ml) for 24 h prior to the assay, and
quantitated by solubilizing the cells in 2 ml of 0.5 N NaOH, 1% SDS, adding 15
ml of Aquasol II, and counting in a scintillation counter. Determinations for both
methodologies were done in triplicate.
To determine if binding to the protein substrate was specific for the protein,
affinity purified antilaminin or antifibronectin (50 to 100/tg/plate) was added
with ceils in the assay. To determine if new protein synthesis was required for
binding to occur, cells were incubated in medium containing 10 to 25 #g/ml
cycloheximide for 4 h prior to the assay and during the assay,
Cell Morphology on Protein-coated Substrates: The morphology of adherent cells was evaluated by performing adhesion as described
above, using substrates coated with 10/~g of laminin or fibronectin per plate,
followed by fixation of the adherent cells with 1% glutaraldehyde in PBS for 30
min. Adherent cells were classified into one of three groups: round, spread, or
curved and elongated. A total of 200 cells were classified on each substrate.
Immunofluorescence on Sciatic Nerve: To evaluate the presence of laminin and fibronectin in vivo, we performed immunofluorescent
localizations on sections of mouse sciatic nerve. Adult BALB/c mice were killed
by cervical dislocation; and the sciatic nerves of both hind limbs removed by
dissection. The nerves were fixed in cold ethanol, dehydrated, and embedded in
paraffin following the procedure of Sainte-Marie (41). Several nerves were fixed
in 2.5% glutaraldehyde in PBS for 2 h, dehydrated through ethanols and
propylene oxide, and embedded in Epon 812 for histological staining with
hematoxylin and eosin, or Linie's Allochrome stain (42). Sections were deparaffmized, hydrated to PBS, and reacted with primary antiserum or pre-immune
rabbit serum 0:20 dilution). The reaction was localized with fluorescein or
rhodamine-conjugated goat anti rabbit IgG (Cappel Laboratories). Sections were
viewed with an Olympus BH fluorescence microscope with epi-illumination using
a 40x oil immersion lens. Photos were taken on Kodak Ektachrome 400 using 2to 3-min exposures.
Published May 1, 1983
kdaltons and weaker bands at 400, 200, and 150 kdaltons (Fig.
2 c, lane 3). The 400- and 200-kdalton bands may be monomers
or intermediate complexes of laminin chains prior to complexing to the 850-kdalton form. No detectable levels of fibronectin
were found in the solubilized cell layer (Fig. 2 c, lane 2). As
before, normal rabbit serum did not precipitate any protein
(Fig. 2 b, lane 1). The presence of the 850- and 150-kdalton
bands from conditioned medium under nonreducing conditions
suggests that these proteins are associated by noncovalent
interactions, or that the 150-kdalton protein is a fragment of
laminin that is recognized by antibodies to the intact laminin
molecule.
Immunocytochemistry
Cell-binding
Assay
The results of one cell-binding assay using the Coulter
Counter for quantitation are presented in Fig. 4. This is representative of a series of five assays using both radio-labeled
and nonlabeled cells. This particular assay used plates coated
with 1, 10, or 20/~g of protein. The amount of protein binding
to plates at various concentrations is given in Table I. Onetailed Student's t test for the difference of two independent
means was performed to determine if cell binding to lamininor fibronectin-coated plates was significantly greater than binding to noncoated plates. In the above assay, binding was
significantly greater to laminin at all three concentrations (1,
10, and 20/~g/plate) than to control plates (P < 0.01 in all
cases). Binding was greater to fibronectin at 20/zg/plate than
binding to control plates (0.025 < P < 0.05), but not at 10 or
I /~g/plate (P < 0.05). Binding to laminin was significantly
greater than binding to fibronectin at all levels (P < 0.01 for 1
and 10 #g, 0.01 < P < 0.025 for 20 #g/plate, one-tailed
Student's t test). Comparison of the data from the five assays
performed, using the values for the 10/xg/plate samples, shows
that laminin significantly improved binding compared with
noncoated plates (P < 0.01 for 4 assays, 0.01 < P < 0.05 for
the fifth assay). In three of the five assays, fibronectin also
provided a better substrate than control plates (0.01 < P <
0.05). However, in three of the assays, laminin was very significantly better than fibronectin at promoting adhesion (P <
0.01), and in no case was fibronectin significantly better than
binding to laminin. The binding to laminin and fibronectin
was inhibited by adding afffmity purified antibodies with the
cells during the incubation period, but was not blocked by
treatment of the cells with cycloheximide (data not shown).
FIGURE 3 Electron micrographs of extracellular immunochemical
staining of RN22F Schwannoma cells. (a) Antilaminin antiserum, x
12,000. (b) Pre-immune rabbit serum, x 11,000. (c) Antifibronectin
antiserum, x 12,000.
PALMAND FURCIIT Larnininand Fibronectin in SchwannomaCells
1221
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The RN22F ceils were immuuochemically stained using
rabbit antilaminin and antifibronectin antisera in the unlabeled
antibody immunoperoxidase technique. Pre-immune rabbit serum and goat serum were used as controls for nonspecific
staining by the primary and secondary antisera, respectively.
Laminin antiserum produced staining along the periphery of
the cells (Fig. 3 a). No staining was observed on or around
ceils of the control samples (Fig. 3 b). Staining with antifibronectin was sparse along the cell periphery, with large areas of
no staining (Fig. 3 c). Some of the small amount of fibronectin
staining seen by electron microscopy may be due to fibronectin
from the growth medium adhering to Schwannoma cell surfaces. As the fibronectin in the growth medium was not radiolabeled, it would not be detected by immunoprecipitation with
autoradiography.
Published May 1, 1983
EO
l
I Laminin
'
.
~
.
.
//
Fibronectin
.
.
//3
g~
f/
,~- j
+,,.g
Io,~
2o,,.g
PROT[IN COATED ON PLATE
FIGURE 4 Results of cell-binding assay using n o n r a d i o - l a b e l e d cells
and Coulter counter. Dashed line represents percent of cells applied
that bind to the noncoated, bacteriological plastic control plate.
Percent of cells b o u n d = ( n u m b e r o f cells b o u n d per 10 s cells
applied) x 100%.
Protein
Laminin
1/Lg
10 p,g
100 gg
Fibronectin
1 gg
10 gg
100/tg
cpm Available*
cpm Bound
Estimated/~g
protein
bound:l:
2.3 x 105
5.5 x 106
--§
8.0 x 103
9.3 x 104
5.2 x 106
0.3
1.7
+10-2011
4.0 x 103
8.1 x 104
3.2 x 105
2.0 x 10 a
1.9 x 104
5.8 x 104
0.5
2.3
17.9
* Counts per minute in 1 ml of protein solution of given concentration.
~: Protein bound estimated by: ~g protein bound = (cpm bound/cpm available) x p.g available.
§ Counts exceeded machine capacity.
11Value estimated using counts available for 1 and 10#g
Cell Morphology on Protein-coated Substrates
During the cell binding assay, we noticed that many ceils
plated on laminin adopted a different, somewhat crescent or
curved, morphology as compared with cells plated on fibronectin or noncoated plastic. The typical cell morphology on
noncoated plastic was round and nonspread (Fig. 5 a). These
cells were bound to the plastic, i.e., they were not removed by
the buffer wash, but they did not spread, a result similar to
that seen for many cell types on plastic that is not treated for
tissue culture. On fibronectin coated plates, a number of cells
spread on the substrate, as seen in Fig. 5 b. However, on
laminin, many cells adopted a curved morphology, as shown
in Fig. 5 c. Curved cells were only very rarely seen on the other
substrates. The results of the classification of 200 cells by
morphology on each of the three substrates (noncoated, laminin
10 ~tg/plate, fibronectin 10/tg/plate) are given in Table II. The
data were analyzed as a 3 x 3 contigency table (44, 45). In
Table II, the first value listed in each cross-category is the
number of cells observed in that category (OBS). The second
value is the expected number of cells (EXP) in that category
1222
the JOURNAL OF CELL BIOLOGY • VOLUME 96, 1983
Sciatic Nerve Staining
Immunofluorescent staining of mouse sciatic nerve was performed to determine if laminin and fibronectin are present in
peripheral nerve in vivo, as the results with the Schwannoma
cell line might indicate. Sections were stained with rabbit
antilaminin or antifibronectin, with preimmune rabbit serum
as the specificity control. Fig. 6 a is a micrograph of a l-gm
resin section of sciatic nerve stained with Lillie's Allochrome
Stain. This stain has a periodic acid-Schiffs reagent step which
stains basement membranes/glycosaminoglycans pink, and a
step which stains collagens blue. In these sections the endoneurium (E) stained pinkish purple, indicating the location of
basement membrane, and the perineurinm (P) stained deeper
purple, indicating a stronger presence of collagens. Fig. 6 b is
a 6 #m paraffin section of sciatic nerve fLxed in cold ethanol
and stained with Lillie's Allochrome. Although the histology
is not as well preserved in the cold ethanol-fLxed tissue, the
structures are recognizable and stain identically to the glutaraldehyde-fixed tissues. Immunofluorescent staining was performed on sections adjacent to that shown in Fig. 6 b. Fig. 6 c
shows a control section treated with preimmune rabbit serum.
Endoneurium (E) and perineurium (P) are indicated. There is
little background staining. Fig. 6 d is a phase-contrast micrograph of the section shown in Fig. 6 c. (Different objectives
had to be used for the phase and fluorescence pictures, so the
field sizes are not identical.) Rabbit antilaminin stained in a
very distinct band in the endoneurium around nerve fibers and
in the perineurium (Fig. 6 e). In contrast to laminin, there is
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TABLE I
Protein Bound to Plates
based on a model of independence--in other words, the number of cells one would expect to be in a given category if cell
morphology were independent of the substrate the ceils were
plated on. (The expected value is calculated as EXP = (row
total) x (column total)/N, where N is the total sample size).
The third value in each category (DIFF) is the difference
between the observed number of cells and the expected number
of cells in the category (OBS - EXP). These differences were
used in the calculation of the X2 statistic (X2 = ~[(OBS EXP)2/EXP] with 4 degrees of freedom). The calculated X2
statistic was 350 (P < 0.005). The observed number of cells in
each category was significantly different from the expected
number of cells if substrate and cell morphology were independent of each other. Examining the differences between the
observed and expected values gives some indication of where
the greatest differences occur. In the first row of Table II (no
coating), it is seen that there are two times more cells in the
round category than expected, and fewer cells than expected in
the spread and curved categories. For the laminin row, the
differences are reversed; there are many fewer cells in the
round category than expected, and more cells in the curved
and spread categories. In the fibronectin row, observed values
for round cells are near expected values, but there are more
ceils in the spread category and fewer cells in the curved
category than expected. To summarize the data: The majority
of the cells binding to the noncoated bacterial plastic do not
spread on the substrate. When the surface is coated with
fibronectin, the cells are able to spread better than on a
noncoated substrate, and do so in a uniform "fried egg" shape
rather than a curved shape. When laminin is used as the
substrate, virtually none of the cells stay in the round nonspread
morphology; two thirds of the cells take on a uniformly spread
morphology, while the other third take on the curved, crescentshaped morphology.
Published May 1, 1983
FIGURE 5 Cell morphology of adherent RN22F Schwannoma cells on protein-coated substrates. Phase contrast. (a) Round cells;
noncoated plastic substrate. (b) Spread cells; fibronectin-coated substrate. (c) Curved and elongated cells; [aminin-coated
substrate, x 400.
TABLE II
Cross Tabular Analysis o[ Cell Morphology on
Coated Substrates
Morphology
Row
Coating
No coating
Fibronectin, 10#g
Column total
Spread Curved
OBS
EXP
171
85.6
28
93.6
I
20,6
DIFF
+85.4
-65.6
-19.6
134
61
total
200
OBS
5
EXP
85.6
93.6
20.6
200
DIFF
-80.6
+40.4
+40.4
OBS
EXP
DIFF
81
85.6
-4.6
119
93.6
+25.4
0
20,6
-20.6
200
257
281
62
600
Cells (1 x 105/plate) were incubated on protein-coated 35-mm bacteriological
plastic plates for 90 rain. Nonbound cells were washed away, and bound cells
fixed with glutaraldehyde. A total of 200 cells from each plate were classified
by morphology. OBS. observed number of cells in category. EXP, expected
number of cells in category (see calculating formula in text}. DIEF, difference
between the observed and expected number of cells (OBS - EXP).
little staining of the endoneurium with antifibronectin, but the
perineudum stains very strongly (Fig. 6 f ) . The antifibronectin
staining that is seen in the endoneurium is very diffuse as
compared with the very distinct bands seen with antilaminin
antiserum. The location of laminin and fibronectin m vivo
appear to correlate with the behavior of the Schwannoma cells
on laminin and fibronectin coated substrates. The Schwannoma cells bind better to laminin in vitro, and in vivo laminin
is close to Schwann ceils, suggesting its production by, or
association with, these cells.
DISCUSSION
Our study shows that a rat Schwannoma cell line, RN22F,
produces both laminin and fibronectin as detected by immunoprecipitation. The laminin appears in immunoprecipitates of
solubilized cell layer and conditioned medium. In immune
precipitates using antilaminin a 150-kdalton band is precipitated along with a 200-kdalton reduced laminin band. Under
nonreducing conditions the 150-kdalton band can be separated
from the 850-kdalton nonreduced laminin. Antifibronectin
PALM AND FUItCHI
Laminin and Fibronectin in Schwannoma Ceils
1223
Downloaded from on June 17, 2017
Laminin, lO/~g
Round
antiserum precipitates a band of 220 kdaltons (reduced) from
conditioned medium. Under nonreducing conditions there are
bands of 440 and 220-kdalton. The 440-kdalton band co-migrates with human plasma fibronectin run on gels in the acetic
acid sample buffer, and the 220-kdalton band co-migrates with
reduced human plasma fibronectin. Fibronectin was not detected in immunoprecipitates of the cell layers suggesting that
it is largely shed from the cells.
Immunocytochemical localization oflaminin and fibronectin
at the electron microscope level indicates that laminin is distributed along the surface of the Schwannoma cells in culture.
Fibronectin is sparsely distributed along the cell surface. As
indicated by immunoprecipitation, almost all of the fibronectin
produced by the Schwannoma cells is released into the culture
medium.
The cell binding assays show that the Schwannoma cells
bind better to laminin than to fibronectin or noncoated bacteriological plastic. Classification of cells by their morphology on
the different substrates indicated that the cells will spread on
a fibronectin substrate, but more will spread on laminin, and
a large number of the cells undergo a change in morphology to
a curved-crescent shape on laminin. The change in morphology
was similar to a morphological change observed when RN22
cells were treated with cyclic nucleotide analogs (35). Finally,
by immunofluorescence, it was found that laminin and fibronectin are present in sciatic nerve. The laminin was localized
in the endoneurium and perineurium, while the fibronectin
was found mainly in the perineurium.
The 150-kdalton band, seen in immunoprecipitates with
antilaminin appears to be a protein chain that is not covalently
linked into the laminin complex. Because of the 150,000 mol
wt o f this protein, it was thought that the protein might be
entactin, a 158,000 lamimn-associated sulfated protein described by Carlin et al. (9). However, antiserum to entactin did
not precipitate this 150-kdalton band in immunoprecipitation
experiments. In contrast to laminin from the EHS tumor, the
laminin produced by the RN22F cells does not appear to have
a 400,000 subunit under reducing conditions, although the total
molecular weight of the nonreduced complex is similar to the
weight of nonreduced EHS laminin. Preliminary work is beginning to indicate that the 150-kdalton band may be a degradation product or subunit gene product that contributes to
the 400-kdalton chain, with degradation of the 400-kdalton
chain occurring at a fast enough rate that the 400-kdalton
product cannot be detected by our method. This is based on
three sets of observations: (a) The RN2 rat Schwannoma line,
a cell line related to the RN22F line used here, produces 400-
Published May 1, 1983
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FiGure 6 I m m u n o f l u o r e s c e n t staining of mouse sciatic nerve. (E) Endaoneurium. (P) Perineurium. (a) Resin section (1/~m) of
mouse sciatic nerve fixed with glutaraldehyde and stained with Lillie's AIIochrome stain, x 1,200. (b) Paraffin section (6/lm) of
mouse sciatic nerve fixed with cold ethanol and stained with Lillie's A l l o c h r o m e stain, x 1,200. (c) I m m u n o c y t o c h e m i c a l control,
stained with p r e - i m m u n e rabbit serum. Paraffin section of cold ethanol-fixed sciatic nerve, x 1,200. ( d ) Phase-contrast micrograph
of the section shown in c. x 900. (e) I m m u n o f l u o r e s c e n t staining with antilaminin antiserum. Paraffin section of cold ethanol-fixed
sciatic nerve, x 1,200. ( f ) I m m u n o f l u o r e s c e n t staining with antifibronectin antiserum. Paraffin section o f cold ethanol-fixed sciatic
nerve, x 1,200.
1224
THE JOURNAL OF CELL BIOLOGY-VOLUME 96, 1983
Published May 1, 1983
cells and basement membrane of the sciatic nerve--appeared
to allow expression of regenerative capacity in the neurons that
is normally not expressed. The bridging of the gap between
severed nerve ends is important in regeneration. When the
sheath surrounding a nerve is destroyed, regeneration of the
nerve takes the form of a neuroma; the axons do not regenerate
and connect properly. Molander et al. (54) have found that a
tube or bridge made of polyglactin 910, a synthetic mesh,
helped guide axon regeneration. In the mesh tube a new nerve
sheath formed as the mesh was absorbed. There was less
tendency for neuroma formation in nerves surrounded by the
polyglactin sheath. Basement membrane proteins such as laminin and fibronectin could play a role by binding to the mesh
and acting as guides. The disorganized proliferation of
Schwann cells around a nerve stump that usually occurs could
be presenting a confusing pattern of basement membranes to
the axon, leading to the sprouting of the neuroma.
Proper regeneration and development of nerves requires
two-way interactions between Schwann cells and neurons. To
produce a basement membrane, Schwann cells need some sort
of signal from axons (3). The signal may be provided by neurite
membrane, as a neurite membrane fraction has been found to
be mitogenic for Schwann cells (55, 56). The signal may be
rather subtle, as Carey and Bunge (57) found that Schwann
cells in culture, with and without neurons present, released
similar patterns of proteins even though Schwann cells in
culture alone do not form a discrete basement membrane. The
interaction of proteins in the presence of neurons appears to be
altered so the proteins organize into the basement membrane
structure.
The presence of a connective tissue matrix (collagens, other
fibroblast-produced proteins) may play a role in development
and proper interaction of Schwann cells and nerves. Bunge
and Bunge (58) found that fascicles of Schwann cells and
neurites that were suspended in culture without contact with a
connective tissue matrix developed abnormally. When a collagen substrate was added on top of the suspended fascicle so
contact was made, development proceeded normally, and the
larger axons in the culture were myelinated properly.
In summary, we found that a Schwannoma cell line produced
laminin and fibronectin. The cultured cells have laminin on
their surface, but release most of the fibronectin into the
medium. The cells preferentially bind to, and change their
morphology in response to a laminin-coated substrate when
compared with fibronectin or noncoated bacteriological plastic.
The localization of laminin, but very little fibronectin, in the
endoneurium of mouse sciatic nerve indicates that laminin
occurs in vivo in a position where it could play a role in the
regeneration and myelination of injured axons. Localization of
fibronectin in the perineurium indicates that fibronectin is
available tO play a role in the Schwann-neuron-connective
tissue interactions described by Bunge and Bunge (58). The
RN22F cells, as a model of Schwann cells, may give us a basic
picture of the role of laminin and fibronectin in the peripheral
nervous system. Future examination of primary Schwann cells
in vitro, and embryonic tissue in vivo may give us a clearer
understanding of the roles of these proteins in the development
and regeneration of the nervous system.
We would like to thank Dr. James McCarthy for his discussions and
ideas on this research, and Elaine Oberle for her help in preparing
photographs for the paper.
This research was supported by grants CA29995 and CA21463,
PALM AND FURCHT Laminin and Fibronectin in 5chwannoma Cells
1225
Downloaded from on June 17, 2017
and 200-kdalton chains, but no 150-kdalton chain using the
same antiserum as used in this study (data not shown). (b)
Western blot analysis using polyclonal rabbit antilaminin antiserum indicates that our antiserum reacts with a faint 150kdalton band as well as the major 400- and 200-kdalton bands
in laminin preparations (data not shown). Immunoprecipitation experiments using this polyclonal antiserum, and a monoclonal rat anti-mouse laminin antibody preparation precipitate
similar patterns of bands from crude laminin preparations,
indicating that the 150-kdalton bands may be part of a laminin
chain. (c) The 150-kdalton band does not seem to be one of
the other major basement membrane components in that it is
not precipitated by anti-entactin antiserum, and it is precipitated by an antilaminin antiserum that has been affinity purified over columns of type IV collagen, fibronectin, and basement membrane proteoglycan to remove contaminating antibodies. This is all circumstantial evidence; further work, ineluding peptide mapping and amino acid analysis, will be
needed to positively identify the 150-kdalton band produced
by the RN22F ceils.
The production of laminin and fibronectin by the RN22F
cell line is consistent with studies by other groups. Kurkinen
and Alitalo (46) have shown that a related Schwannoma cell
line, RN2, produces fibronectin and also a procollagen of
unknown type. The localization oflaminin in the endoneurium
and fibronectin in the perineurium generally agrees with the
distribution of collagens in the peripheral nervous system.
Shellswell et al. (47) found types IV and V collagen in the
basement membrane portion of the endoneurium, and types I
and III collagen in the perineurium and extra-basement membrane regions of the endoneurium. Laminin is often associated
with type IV collagen in basement membranes, while fibronectin is often found in the presence of types I and III collagen.
There are two specific times when the production of laminin
and fibronectin by Schwann cells may be important in the
peripheral nervous system: during development, basement
membranes may be important in guiding developing axons to
their final location and in promoting myelination; and following injury to nerves, the basement membrane proteins produced by the Schwann cells may be important in stimulating
and guiding the sprouting neurites so they can properly regenerate and reestablish the disrupted nerve pathway.
There is evidence accumulating to support a role for basement membrane proteins in nerve development. Nonneuronal
cells in culture make a "substrate conditioning factor" that
appears to guide elongating nerve fibers in vitro (48). Akers et
al. (49) found that pre-treatment of growth substrate with
fibronectin promoted retinal neurite outgrowth (49). Manthorpe et al. (50) reported that conditioned medium from
RN22F Schwannoma cultures contains a factor that promotes
neurite outgrowth. Fibronectin and collagen appear to play a
role in neural crest migration in early embryos (51, 52).
Schwann cells and their products may be vital in the proper
regeneration of injured nerves. From early studies it has been
known that nerves in the peripheral nervous system had the
capacity to regenerate, but central nervous system neurons
rarely, if ever, regenerated (4). Recently, Aguayo et al. (53)
reported that an autologous graft of sciatic nerve between lower
cervical or upper thoracic spinal cord and medulla oblongata
allowed spinal and brain stem axons to extend further than
they usually grow in intact animals (without grafts). The change
of the neurological environment from central nervous system
to that of the peripheral nervous system--with the Schwann
Published May 1, 1983
National Institutes of Health, National Cancer Institute (NIH/NCI),
and by the Leukemia Task Force. Dr. Furcht is the recipient of a Stone
Professorship in Pathology, University of Minnesota, and a Research
Career Development Award, CA00651, from NIH/NCI.
This paper, and work reported, partially satisfies the doctoral thesis
requirements for Sally L. Palm. Portions of the work were presented in
abstract form at the 1982 meeting of the American Society for Cell
Biology.
Received for publication 19 July 1982, and in revised form 10 January
1983.
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TH~ IOURNAL OF CELL BIOLOGY. VOLUME 96, 1983
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