©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inter--inhibitor Is Required for the Formation of the Hyaluronan-containing Coat on Fibroblasts and Mesothelial Cells (*)

Anna Blom(§), HPertoft, and Erik Fries

From the (1) Department of Medical and Physiological Chemistry, University of Uppsala, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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--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.


INTRODUCTION

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--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 (II),() 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 II isolated from human serum will support the formation of the hyaluronan-containing coats on both fibroblasts and mesothelial cells.


EXPERIMENTAL PROCEDURES

Purification of Inter--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 II (Dakopatts, Glostrup, Denmark). Fractions containing II 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 II 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 II 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.

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 10cells 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 10cells/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 II from Sera Protein A-Sepharose (150 µl; Pharmacia) was suspended in 500 µl of a solution containing 5 mg/ml rabbit antibodies against human II (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 II 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 II-depleted plasma, the concentration of II was at least 15-fold lower than in normal plasma. There was no significant decrease in the II concentration in haptoglobin- or plasminogen-depleted plasma.


RESULTS AND DISCUSSION

For the isolation of human II, 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 II 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 II ( 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 II (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 II. 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 II, 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 II, no coats were seen. In those cases where purified II 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).

In their natural environment, fibroblasts and mesothelial cells are surrounded by interstitial fluid. Here, the level of II 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.

To see if serum components other than II might induce coat formation, we incubated the cells with serum that had been depleted of II. 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 II 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 II (20, 26, 32) .

It has recently been shown that bovine pre--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) .


FOOTNOTES

*
This work was supported by grants from the Swedish Natural Science Research Council (to E. F.) and from the Swedish Medical Research Council (to H. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 46-18-17-41-99; Fax: 46-18-17-49-75; E-mail: fries@bio.embnet.se (for A. B.).

The abbreviations used are: II, inter--inhibitor; PMSF, phenylmethanesulfonyl fluoride; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; EGF, epidermal growth factor.

A. Blom, H. Pertoft, and E. Fries, unpublished observation.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.