From the
Cultured cells of various origins have been shown to be
surrounded by a hyaluronan-containing coat, a structure that can be
visualized by its ability to exclude large particles such as
erythrocytes. When cultured in medium with no or low concentrations of
serum, the cells lose their coats, although they still produce
hyaluronan; upon the addition of serum, the coats are formed again.
Here, we show that the serum protein inter-
Several cell types such as mesothelial cells
(1) ,
chondrocytes
(2, 3) , synovial cells
(4) ,
fibroblasts
(4) , smooth muscle cells
(5) , and certain
tumor cell lines
(6, 7) have been shown to be
surrounded in vitro by a pericellular matrix or coat. These
coats can easily be visualized by their capacity to exclude
erythrocytes
(4) and other particles
(4, 8) .
The polysaccharide hyaluronan (hyaluronic acid) is a major component of
these structures as shown by the fact that they are disrupted upon
treatment with Streptomyces hyaluronidase
(4) . The
function of the coat has not yet been established, but it has been
suggested to protect cells from viral infections
(9) and from
cytotoxic effects of lymphocytes
(8, 10) . It may also
play a role in differentiation
(2, 11) and in
stabilizing the cellular microenvironment
(4) .
At least for
some coat-forming cells, it has been shown that when cultured in medium
with no or low concentrations of serum, they produce normal quantities
of hyaluronan, but do not acquire coats
(1, 12, 13) . This observation indicates that
serum contains a factor that stabilizes the polysaccharide structure
(14) . In recent studies on the hyaluronan-containing
extracellular matrix of cumulus cell-oocyte complexes from mice, two
proteins that stabilized the matrix were isolated from fetal bovine
serum. They were found to belong to the inter-
Purification of Inter-
Normal
human mesothelial cells were obtained from pleural effusions as
described by Versnel et al. (23) and were a gift of
Dr. P. Heldin of this department. These cells were cultured in
Ham's F-10 medium supplemented with 15% FBS, 10 ng/ml epidermal
growth factor (EGF; Sigma), 0.4 µg/ml hydrocortisone, 4 mML-glutamine, and 0.25 mg/ml gentamicin. For all experiments
described in this paper, 3
For the isolation of human I
In their natural environment, fibroblasts and mesothelial cells are
surrounded by interstitial fluid. Here, the level of I
To see if
serum components other than I
It has recently been
shown that bovine pre-
We thank Paraskevi Heldin and Alan McWhirter for
providing cells, Mervi Enojärvi and Nahren Serkes Baba for
technical assistance, and Torvard Laurent and Priit Teder for valuable
discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-inhibitor can replace
whole serum as an inducer of the formation of the coats on fibroblasts
and mesothelial cells. The physiological role of inter-
-inhibitor
has so far been unclear; our findings, together with those obtained
with cumulus cell-oocyte complexes (Chen, L., Mao, S. J., and Larsen,
W. J. (1992) J. Biol. Chem. 267, 12380-12386), suggest
that inter-
-inhibitor and related proteins have a general function
as stabilizers of hyaluronan-containing pericellular coats.
-inhibitor family
(15, 16) , a group of proteins that are composed of
bikunin, which consists of a 16-kDa polypeptide and an 8-kDa
chondroitin sulfate chain, and various combinations of three homologous
heavy chains of 85-96 kDa, called HC1, HC2, and HC3
(17) .
In all these proteins, the polypeptides are covalently linked via the
chondroitin sulfate chain of bikunin
(18) . In human plasma, the
major bikunin-containing protein is inter-
-inhibitor
(I
I),
(
)
which contains HC1 and HC2. The
second most abundant member of this group is pre-
-inhibitor, which
consists of bikunin and HC3
(19, 20) . The corresponding
proteins in bovine plasma contain different combinations of the heavy
chains
(16) . In this paper, we extend the observation made on
cumulus cell-oocyte complexes by showing that I
I isolated from
human serum will support the formation of the hyaluronan-containing
coats on both fibroblasts and mesothelial cells.
-inhibitor Inter-
-inhibitor was isolated from human plasma in four steps. All
preparative work was done at +4 °C, and fractions were kept at
-20 °C when their contents were analyzed.
Step 1: Ammonium Sulfate Precipitation
Citrated human
plasma (100 ml; obtained from the Blood Center of University Hospital,
Uppsala) was brought to 30% saturation with solid ammonium sulfate,
stirred for 30 min, and centrifuged at 10,000 g for 15
min. The supernatant was collected and brought to 50% saturation with
ammonium sulfate and centrifuged. The pellet was dissolved in 6 ml of
Tris-buffered saline (50 mM Tris-HCl, pH 8.0, 150 mM
NaCl) containing 0.2 mM phenylmethanesulfonyl fluoride (PMSF;
Sigma), and the solution was dialyzed against Dulbecco's
phosphate-buffered saline (PBS) lacking calcium and magnesium;
precipitates were removed by centrifugation.
Step 2: Zinc Chelate Chromatography
Chelating
Sepharose Fast Flow (60 ml; Pharmacia, Uppsala) was activated according
to the manufacturer's instructions and equilibrated with PBS
supplemented with 0.2 mM PMSF. Half of the material from the
previous step (0.5 g of protein) was then applied at a flow rate of 60
ml/h, and the column was washed with 120 ml of PBS and 60 ml of PBS
containing 0.8 M NaCl at a flow rate of 100 ml/h. Finally, PBS
with 20 mM EDTA was used to elute II from the column at a
flow rate of 60 ml/h, and 5-ml fractions were collected. The fractions
were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed
by immunoblotting with rabbit antibodies against human I
I
(Dakopatts, Glostrup, Denmark). Fractions containing I
I were
pooled, and the proteins were concentrated by ultrafiltration.
Step 3: Preparative Electrophoresis
Half of the
material from Step 2 (20 mg of protein in 1.0 ml) was mixed with 200
µl of sample buffer and loaded on a polyacrylamide gel (6% T, 2.7%
C; 60 28 mm) in a Model 491 preparative cell (Bio-Rad). The
buffer throughout the system was 0.1 M Tris borate, pH 8.0,
containing 2 mM EDTA and 0.2 mM PMSF. Electrophoresis
was performed at 5 watts of constant power. Elution was done with the
electrode buffer at a flow rate of 1 ml/min, and 10-ml fractions were
collected. Fractions containing I
I were identified as described
above and pooled, and the volume was reduced to 600 µl by
ultrafiltration.
Step 4: Gel Filtration
The material from the
previous step (1 mg of protein in 0.5 ml) was applied to a Superose 12
gel (30 1 cm; Pharmacia) equilibrated with PBS. Chromatography
was performed at a flow rate of 0.2 ml/min, and 0.5-ml fractions were
collected. The fractions were analyzed by SDS-PAGE followed by silver
staining and immunoblotting. The concentration of I
I was
determined from the absorbance at 280 nm ( A
= 0.71
(21) ). Purification of Human Urinary Bikunin Urine of women in the third trimester of pregnancy was obtained from a
mother care center, and bikunin was isolated essentially as described
(22) ; the yield was
3 mg/liter of urine. Cell Culture Conditions Normal human fibroblasts from biopsies of forearm skin from healthy
volunteers were the gift of A. McWhirter of this department and were
cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum (FBS; Life Technologies, Inc.), 4
mML-glutamine, and 0.25 mg/ml gentamicin.
10
cells of both types
were seeded on 35-mm diameter dishes and cultured overnight. Assay of Cell Coat Formation Complete medium was replaced by starvation medium: Dulbecco's
modified Eagle's medium supplemented with glutamine and
gentamicin for fibroblasts and Ham's F-10 medium containing 0.5%
FBS, EGF, hydrocortisone, glutamine, and gentamicin for mesothelial
cells. The cell coats were then removed by the addition of
Streptomyces hyaluronidase (5 units/ml; Seikagaku Koyogo,
Tokyo) followed by a 2-h incubation. The cells were incubated for 16 h
in starvation medium supplemented as described below. The cell coats
were visualized essentially as described
(4, 6) . Horse
erythrocytes were fixed with formaldehyde, washed, and suspended at 4
10
cells/ml in PBS containing 1 mg/ml bovine serum
albumin (Sigma). An aliquot of the erythrocyte suspension (100 µl)
was then added to the culture medium, and the erythrocytes were allowed
to settle for 15 min at 37 °C. The culture dishes were examined
microscopically (Zeiss Axiovert 35), and the number of cells with and
without clouds was scored. Images of randomly selected cells with
clouds were taken with a video camera (Dage MTI ccD72) connected to a
computer (Sun Microsystems Inc.) using the program IC 300 (Inovision
Corp., Durham, NC). The perimeters of the erythrocyte-excluded area and
the area delimited by the plasma membrane were then traced with a
cursor on the computer screen for at least 20 cells, and the size of
the pericellular coats, defined as the ratio between these values, was
determined. A ratio of 1 represents no detectable pericellular matrix. Removal of I
I from Sera Protein A-Sepharose (150 µl; Pharmacia) was suspended in 500 µl
of a solution containing 5 mg/ml rabbit antibodies against human
I
I (Dakopatts), and the test tube was rotated end-over-end
overnight at 4 °C. The gel beads were then washed twice with 500
µl of PBS and resuspended in 500 µl of the same buffer. Normal
human serum (500 µl; obtained from the Blood Center of University
Hospital) was then added, and the suspension was incubated for 5 h.
Finally, the gel beads were removed by centrifugation, and serial
dilutions of the supernatants were analyzed for I
I by SDS-PAGE
followed by immunoblotting. As controls, sera were depleted of
haptoglobin or plasminogen with the respective antibodies obtained from
Dakopatts. The optical densities of the bands on the nitrocellulose
filters were measured by optical scanning. In I
I-depleted plasma,
the concentration of I
I was at least 15-fold lower than in normal
plasma. There was no significant decrease in the I
I concentration
in haptoglobin- or plasminogen-depleted plasma.
I, we used a four-step
procedure that is similar to the one previously described by Salier
et al. (24) , except that preparative gel
electrophoresis was used instead of ion-exchange and hydrophobic
chromatography. From 100 ml of serum, 1.0-1.2 mg of pure protein
was obtained. That the isolated protein was indeed I
I was verified
by its apparent molecular mass upon SDS-PAGE (250-220 kDa;
Fig. 1
, lane 3) and gel filtration
(400-300 kDa; data not shown) and the fact that it reacted with
antibodies against I
I ( lane 4). The purpose of
the last purification step (gel filtration) was mainly to remove
immunoreactive material of low molecular mass, apparently degradation
products. Consistent with previous reports on the instability of
I
I
(21, 25) , we found that the addition of a
protease inhibitor to the buffer solutions increased the yield.
Figure 1:
Isolation of II
from human serum. Human serum was fractionated by ammonium sulfate
precipitation followed by chelating chromatography, preparative gel
electrophoresis, and gel filtration. Lanes 1-3 show the
material obtained from the last three steps analyzed by SDS-PAGE (in 6%
polyacrylamide) followed by silver staining. Lane 4 shows SDS-PAGE of the final product (as shown in lane 3) followed by immunostaining with antibodies against
I
I. The molecular masses of standard proteins run in parallel are
also shown.
Fig. 2
( A and C) shows how the pericellular
coats on mesothelial cells and fibroblasts were detected by the
addition of erythrocytes to the cell cultures. Morphometric analysis
showed that the ratio of the erythrocyte-free area to cell area was
similar for both cell types: 2.5 ± 0.6 for mesothelial cells and
2.2 ± 0.5 for fibroblasts. For the coat formation assay, the
cells were deprived of their coats by hyaluronidase treatment
(Fig. 2, B and D). After a subsequent
incubation for 16 h in starvation medium supplemented with 15% serum,
the relative number of cells with coats was similar for mesothelial
cells and fibroblasts: 42 and 34%, respectively (Fig. 3). In the
absence of serum, 10% of the fibroblasts and 1% of the mesothelial
cells had coats. Fig. 4shows the relative number of fibroblasts
that had acquired coats upon incubation at different serum
concentrations. The pattern obtained suggests that the interaction of
the serum and the cells can be described as a simple binding isotherm.
Indeed, the inset shows that the same data (after subtraction
of the relative number of cells with coats in the absence of serum:
12%) can be fitted to a straight line in a Scatchard plot. This
analysis shows that half-maximal formation of coats was obtained at a
30% final concentration of serum and that 87% (+12%) of the cells
could acquire coats. There was no significant difference in the
relative sizes of the coats at 10, 40, and 80% serum, suggesting that
their formation is an all-or-none type of process. Similar experiments
with mesothelial cells showed that their response to different serum
concentrations resembled that of the fibroblasts.
Figure 2:
Visualization of pericellular coats.
Fibroblasts ( A) and normal human mesothelial cells
( C) were cultured in complete medium. Fixed erythrocytes were
then added to the cultures and allowed to settle. Note that the
erythrocytes are excluded from an area larger than that of the cells.
B and D show a fibroblast and a mesothelial cell,
respectively, whose coats were removed by hyaluronidase treatment
before the erythrocytes were added.
Figure 3:
II requirement for coat formation.
Fibroblasts and mesothelial cells were deprived of their coats by
hyaluronidase treatment and were then incubated in starvation medium
supplemented with the indicated components. Coats were visualized as
described for Fig. 2, and the relative number of cells with coats was
determined. The numbers of experiments are shown in
parentheses, and the error bars show the
standard deviation of the mean. Shaded and open bars show fibroblasts and mesothelial cells,
respectively.
Figure 4:
Serum requirement for coat formation.
Fibroblasts were deprived of their coats by treatment with
hyaluronidase and were then incubated in starvation medium supplemented
with human serum at the indicated concentrations. Coats were visualized
as described for Fig. 2, and the relative number of cells with coats
was determined. Shown are the mean values from two experiments. The
inset shows a Scatchard plot of the same data, except that the
relative number of cells with coats in the absence of serum was
deducted from all values. C and S represent the
relative number of cells with coats and serum concentration,
respectively.
To see if II
could replace human serum as an inducer of coat formation, we incubated
fibroblasts and mesothelial cells in starvation medium containing 70
µg/ml I
I, a concentration corresponding to 12-16% serum
(26, 27) . The relative number of cells that acquired
coats was 40 and 28%, respectively, i.e. close to the values
obtained with 15% serum (Fig. 3). The cellular production of
hyaluronan in mesothelial cells has been shown to be stimulated by EGF
and hydrocortisone
(1) . In agreement with these results, we
found that when these cells were incubated in medium lacking EGF and
hydrocortisone but containing 0.5% FBS and 70 µg/ml I
I, no
coats were seen. In those cases where purified I
I was added to the
medium, albumin (0.5 mg/ml) was present as a stabilizer. Cells
incubated in medium containing albumin only did not form pericellular
coats. Also, bikunin (50 µg/ml) had no effect (data not shown).
I is
probably 15-30% of that in blood plasma
(28, 29, 30) . This fact and the data shown in
Fig. 4
suggest that changes in vascular permeability could affect
the amount of the hyaluronan-containing extracellular matrix in
connective tissue. Interestingly, the concentration of fetal bovine
serum required for half-maximal stabilization of cumulus cell-oocyte
complexes
(13, 15, 31) is at least 100 times
lower than the corresponding value that we have obtained for the
formation of the coats on fibroblasts and mesothelial cells with normal
bovine serum. This difference does not seem to be due to the serum
since in our system, the activity of fetal bovine serum was similar to
that of normal bovine or human serum (30% more efficient).
(
)
It therefore appears that the extracellular matrices are
formed by processes that are, at least in part, different.
I might induce coat formation, we
incubated the cells with serum that had been depleted of I
I.
One-half of the fibroblasts and one-fifth of the mesothelial cells
displayed coats under these conditions compared with normal serum. As a
control, the same cells were incubated with serum depleted of
haptoglobin or plasminogen; the number of cells with coats was
essentially the same as with normal serum. With both cell types, more
cells displayed coats when incubated with serum lacking I
I than
with starvation medium. This difference might be due to a higher
hyaluronan production in the presence of serum and/or the presence of
other coat-inducing serum proteins that are not recognized by the
antibodies. A possible candidate for such a protein is
pre-
-inhibitor, whose concentration in normal human plasma is
20-40% of that of I
I
(20, 26, 32) .
-inhibitor can bind reversibly to hyaluronan
(33) , and it has been proposed that stretches rich in
positively charged amino acid residues might constitute the binding
sites. These findings provide strong evidence that pre-
-inhibitor
and related proteins stabilize the pericellular coats by direct binding
to hyaluronan. However, there is also evidence that the coat contains
other components produced by the cells that contribute to its stability
(13) .
I, inter-
-inhibitor; PMSF, phenylmethanesulfonyl fluoride;
PBS, phosphate-buffered saline; PAGE, polyacrylamide gel
electrophoresis; FBS, fetal bovine serum; EGF, epidermal growth factor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.