©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Myofibroblasts from Scleroderma Skin Synthesize Elevated Levels of Collagen and Tissue Inhibitor of Metalloproteinase (TIMP-1) with Two Forms of TIMP-1 (*)

(Received for publication, July 29, 1994; and in revised form, December 5, 1994)

Theresa Z. Kirk Mina E. Mark Chu C. Chua Balvin H. Chua Maureen D. Mayes (§)

From the Division of Rheumatology, Department of Internal Medicine and the Departments of Pathology and Immunology, Wayne State University School of Medicine, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cultured fibroblasts derived from skin biopsies from scleroderma patients and normal individuals were examined for the presence of smooth muscle alpha-actin, a marker for myofibroblasts. Six of eight scleroderma cell lines were found to be 50% or more positive for alpha-actin while three of four normal lines and one cell line derived from unaffected skin of a scleroderma patient were less than 10% positive. The cultured fibroblasts from affected scleroderma skin were largely myofibroblasts, a phenotype found in biopsies of scleroderma tissue, as well as other fibrotic lesions, wound healing, and tumor desmoplasia. The data support the hypothesis that a certain activated fibroblast phenotype predominates in scleroderma. The activated fibroblast is the myofibroblast. Both collagen and TIMP (tissue inhibitor of metalloproteinases) were elevated in the alpha-actin positive (myofibroblast enriched) cultures. In addition, the myofibroblast-enriched cultures displayed a more prominent TIMP doublet band pattern on SDS-polyacrylamide gel electrophoresis.


INTRODUCTION

Fibroblasts synthesize and maintain the connective tissue matrix and also repair the matrix after injury. A growing body of evidence indicates that fibroblasts are not a homogeneous population but can be distinguished by a number of differences including rates of collagen synthesis (LeRoy, 1974; Needleman et al., 1990; Mayes et al., 1988; Diegelmann, et al., 1979), affinity for C1q (Bordin et al., 1984), prostaglandin E(2) synthesis (Korn, 1983), parathyroid hormone responsiveness (Goldring et al., 1990), glucocorticoid responsiveness (Russell et al., 1989), and expression of some smooth muscle specific proteins (Sappino et al., 1990a; Lazard et al., 1993).

Scleroderma is an autoimmune disease characterized by the dual features of a noninflammatory microvasculopathy and excess collagen accumulation in the skin and internal organs. Botstein et al.(1982) have suggested that the fibrosis seen in scleroderma is the result of the selection of a certain fibroblast phenotype. A number of other lines of evidence are in support of this theory in scleroderma and in other fibrotic disorders.

Smooth muscle alpha-actin-positive fibroblasts (myofibroblasts) are found in scleroderma and in a number of other fibrotic conditions (Sappino et al., 1990b). Ludwicka et al.(1992) have identified myofibroblasts from cultured cells obtained at bronchoalveolar lavage from scleroderma patients. Myofibroblasts also appear transiently in wound healing (Darby et al., 1990). Additionally, cultured fibroblasts from affected skin of scleroderma patients (LeRoy, 1974), from keloids (Uitto et al., 1985), and from phenytoin-induced gingival hyperplasia (Hassell et al., 1976) all continue to exhibit an abnormal phenotype even after several passages in culture. Lastly, in situ hybridization studies show that not all but rather certain distinguishable fibroblasts in keloids (Peltonen et al., 1991) and in affected scleroderma skin (Kahari et al., 1988) contain high levels of collagen message. The possibility that the myofibroblast phenotype might correspond to this ``activated fibroblast'' phenotype found in fibrosis has thus far not been reported.

Earlier data (Mayes and Kirk, 1991) indicated that some scleroderma cell lines synthesize elevated levels of TIMP. (^1)High levels of TIMP could play a role in the fibrotic process by inhibiting collagen degradation. Qualitative differences in TIMP synthesis were also observed. Immunoprecipitates of TIMP from scleroderma cells appeared as a doublet on gel electrophoresis while the TIMP immunoprecipitates from normal cells appeared largely in one band.

In this study, cultured dermal fibroblasts derived from scleroderma patients and normal individuals were examined for evidence of the myofibroblast phenotype and relative synthetic rates of collagen and TIMP. TIMP synthesis was further characterized by resolution of the two forms by SDS-gel electrophoresis.


EXPERIMENTAL PROCEDURES

Patient Selection

All scleroderma patients met the American Rheumatology Association (now American College of Rheumatology) criteria for the diagnosis of scleroderma (Masi et al., 1980). Ten patients had diffuse disease, and one had limited disease. All were antinuclear antibody positive and had no evidence of mixed connective tissue disease. All but one patient had biopsies from the affected skin of the forearm. One patient with skin involvement limited to the fingers (sclerodactyly) had a biopsy taken from unaffected forearm skin.

Cell Culture

Fibroblasts were grown out from explants of the skin biopsies of scleroderma patients and normal volunteers. Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.). Penicillin (100 IU/ml) and streptomycin (100 µg/ml) were included in the growth media. Cells were passaged by trypsinization (0.05% trypsin, 0.53 mM EDTA tetrasodium salt (4Na)) and the routine passage was 1:2. Cells were generally taken for experiments between passages five and 10 unless otherwise noted.

Preparation of TIMP Antisera

Five mg of human TIMP was kindly provided by Synergen Corporation (Denver, CO). An aliquot of inhibitor (100 µg) was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into rabbits at multiple sites. Booster injections of 50 µg of TIMP were given every 2 weeks. The specificity of the antibody was checked by Western blot analysis.

[S]Methionine Labeling and Immunoprecipitation of Labeled TIMP

Confluent cells in 12-well plates were labeled with 10 µCi/ml [S]methionine in methionine-free DMEM with 5% dialyzed serum. After 24 h, the medium was collected and frozen at -60 °C until further use. Total [S]methionine incorporation into media proteins was measured by 10% trichloroacetic acid precipitation, and this value was used to normalize TIMP-1 counts in gel electrophoresis in some experiments.

For routine immunoprecipitation, 150 µl of labeled medium were combined with 40 µl of antibody and 100 µl of incubation buffer. The polyclonal antisera against human TIMP were prepared as described above. Additional rabbit antiserum was kindly provided by Dr. Henning Birkedal-Hansen, University of Alabama at Birmingham. The incubation buffer consisted of 100 mM NaCl, 50 mM Tris-HCl (pH 7.2), 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.15% SDS, and 0.01% sodium azide. The medium and antibody were incubated at 37 °C for 2 h. Protein A-Sepharose (150 µl of a 1 g/10 ml solution in incubation buffer) was then added, and the incubation was continued for an additional 1 h at room temperature.

The immunoprecipitate was centrifuged in a microcentrifuge and washed six times in wash buffer (0.16 M NaCl, 0.5% Triton X-100, 0.03% bovine serum albumin, 0.1% SDS, 0.01% sodium azide). The immunoprecipitate was then heated in 80 µl of gel sample buffer for 5 min at 95 °C, cooled, and centrifuged to remove the Protein A-agarose beads. Fifty µl of the supernatant was taken for gel electrophoresis. Gel electrophoresis and sample buffer were according to Laemmli(1970), and gels were processed for fluorography following the method of Skinner and Griswold(1983). Intensity of the fluorographic bands was quantitated with an AMBIS 4000 Optical Imaging System (AMBIS, San Diego, CA). In some experiments the radioactivity in the gel was quantitated directly using the AMBIS Radioanalytical Imaging System.

Collagen Measurement

Confluent cultures of fibroblasts in 12-well plates were labeled with 10 µCi/ml [^3H]proline for 24 h in either serum-free DMEM or DMEM with 10% serum. The labeling medium was supplemented with 50 µg/ml ascorbate and 50 µg/ml beta-aminoproprionitrile. Labeled media were collected and frozen at -60 °C until analysis. Collagen synthesis was estimated by pepsin digestion and salt precipitation of the media following the method of Webster and Harvey(1979). Total [^3H]proline incorporation into media proteins was measured by 10% trichloroacetic acid precipitation.

DNA Measurement

Cell monolayers in 12-well plates were fixed with 5% trichloroacetic acid and kept frozen until DNA was measured spectrophotometrically by the method of Burton(1956).

Immunofluorescence

For immunofluorescence cells were grown on coverslips and placed in serum-free medium for 2 days prior to fixation with methanol. Fixed cells were first incubated with 20 µl of fetal calf serum for 30 min and rinsed in phosphate-buffered saline. Monoclonal antibody to smooth muscle alpha-actin (15-20 µl of a 5 µg/ml solution, Boehringer Mannheim), was then added and rinsed off with phosphate-buffered saline 1 h later. The coverslip was then incubated with 20 µl of a 1:10 dilution of fluorescein (DTAF)-conjugated AffiniPure F(ab`)2 Fragment goat anti-mouse IgG (Jackson Immunoresearch Laboratories) and rinsed with phosphate-buffered saline. Cells were examined with a Zeiss Axiophot microscope. The number of positive cells and total cells were counted in defined fields on the coverslip. Cells were considered positive for alpha-actin if actin fibers were clearly outlined by the immunostaining procedure.

Statistical Analysis

Student's t test was used to analyze differences between means obtained from two groups.


RESULTS

alpha-Actin Staining

Indirect immunostaining of cells for smooth muscle alpha-actin showed that some fibroblast cultures did stain positively as shown in Fig. 1, panel A. Although the cells did not stain as brightly as rat heart smooth muscle cells used as a positive control (data not shown), the staining was specific in that it did clearly outline actin fibers in the cell. Other cell lines were predominately negative as shown in Fig. 1, panel B. There was some background or autofluorescence in these negative cultures, but it was not possible to delineate actin fibers.


Figure 1: Panel A shows the typical staining pattern observed for alpha-actin-positive cultures. Actin fibers are clearly outlined in the majority of cells. Panel B shows the typical staining pattern for cultures which were predominately alpha-actin negative. A few actin fibers can be seen in some cells, but the majority of cells do not show positive fluorescence. Magnification is times125. Both fields were photographed under the same conditions, and the photographic negatives were also treated and printed identically.



Essentially all cultures were predominately positive or negative for alpha-actin although both cell types were evident in all cultures, and one cell line (S6) appeared to be a 50:50 mixture of the cell types. An estimate of the number of positive cells in a sample is summarized in Table 1. Six of eight scleroderma cell lines derived from affected skin were 50% or more positive. Fibroblasts from unaffected skin of one scleroderma patient and three normal fibroblast lines were predominately negative. Surprisingly one of the normal cell lines(N3) was largely positive for alpha-actin.



Variable Stability of Fibroblast Phenotypes

Some cell lines in culture that were initially found to be predominately one phenotype in an early passage were found to be predominately another phenotype a few passages later. For example, line N3 consisted primarily of myofibroblasts at passage four, but at passage eight only a few myofibroblasts were seen. On the other hand, line N4 was predominantly smooth muscle alpha-actin negative at passage four yet myofibroblasts were the dominant fibroblast phenotype at passage eight. In other cases, the phenotype appeared relatively constant as judged by smooth muscle alpha-actin staining or relative rate of collagen synthesis (data not shown).

Collagen Synthesis Measurements

Collagen synthesis was compared in two groups. The first group was labeled with [^3H]proline in the absence of serum and normalized by determining total [^3H]proline incorporated into cell media protein. As seen in Table 2, S2 and S3, two scleroderma cell lines, had the greatest counts in pepsin-resistant protein, as well as the highest ratios of pepsin-resistant counts/total incorporated counts in the media. When the data were normalized these two cell lines were clearly two to three times higher in collagen synthetic activity than the other cell lines used in this sampling. The one scleroderma cell line in this group (S9) that did not display elevated synthesis of collagen was also alpha-actin negative.



The second group was labeled in the presence of 10% serum. Preliminary data indicated that elevated TIMP synthesis correlated with elevated collagen synthesis. Serum was added to reduce the possibility that metalloproteinases might be active in the low TIMP media either during the labeling period or before the pepsin digestion step in the collagen assay. The data in this second group were normalized with respect to total DNA. Although pepsin-resistant counts were high for a number of cell lines (Table 2), when normalized the high collagen producers were N3, an alpha-actin-positive cell line derived from normal skin, and one alpha-actin-positive scleroderma cell lines (S1). The cell line from unaffected skin of a scleroderma patient and two affected scleroderma cell lines (one alpha-actin positive and the other 50:50 alpha-actin positive) were low collagen producers. Again the normalized pepsin-resistant counts showed a clear separation of high and low collagen producers.

Taken together, the collagen data show that all four of the alpha-actin-negative cell lines examined were relatively low collagen producers. Four out of the six alpha-actin positive cell lines synthesized high levels of collagen.

Two additional affected scleroderma (S10 and S11) and two normal cell lines (N5 and N6) were evaluated for collagen but not for alpha-actin staining. Both normal cell lines and one scleroderma cell line (S10) were low collagen producers (pepsin-resistant counts/minute divided by 10% trichloroacetic acid precipitate counts/min = 0.18, 0.17, and 0.08, respectively). The other scleroderma cell line S11 was the highest collagen producer of the cell lines tested, with pepsin-resistant counts/min = 22,510 and pepsin-resistant counts/min divided by µg of DNA = 12,037.

TIMP Quantitation

TIMP synthesis was also measured in two groups. Fluorography of the immunoprecipitated TIMP bands on 10% non-reducing gels are shown in Fig. 2. The immunoprecipitate could be resolved into a doublet. The same result was seen with two different preparations of antibody from two different sources. The major band is approximately 28 kDa, and the minor band appears to be approximately 30 kDa. The relative amounts of the two TIMP bands (i.e. the ratio of the 30 kDa TIMP-1 band/28 kDa TIMP-1 band) seen in an individual cell line as well as the relative amounts of total TIMP produced by different cell lines are summarized in Table 3. The data for total TIMP production did not show a clear separation of high and low producers. The two lowest values included one alpha-actin-positive and one alpha-actin-negative line. Cell line S8 (alpha-actin negative) showed an intermediate value. Two positive cell lines and one negative cell line showed high levels. One positive cell line (S6) was clearly higher than all the rest.


Figure 2: Electrophoretic separation of TIMP-1 immunoprecipitates on 10 times non-reducing SDS-polyacrylamide gels. The TIMP-1 doublet is more readily seen in N3, S6, S1, S3, and S2. Densitometric quantitation of the fluorographs is shown in Table 3.





The second group of cells in the TIMP quantitation involved cell lines at high passage (passage 19-25). Two scleroderma cell lines, S3 and S2, were found to be relatively high TIMP producers while N2 was intermediate, and line N5 was a low TIMP producer. Lines S3 and S2 are scleroderma- and alpha-actin-positive fibroblasts. Cell line N2 was alpha-actin negative and cell line N5 was not stained for alpha-actin.

Combining the TIMP and alpha-actin data it was found that five of the alpha-actin-positive cell lines had relatively high levels of TIMP in the media while one had low TIMP. One alpha-actin-negative cell line had a high TIMP level. Two negative cell lines were in the intermediate range, and one had a very low level. On the whole, alpha-actin-positive cells were likely to have higher TIMP levels.

Densitometry data were compared with direct quantitation of counts from the gel. This is included in Table 3. In this analysis both TIMP-1 bands were combined because of the difficulty in separating the two when measuring the radioactivity directly from the gel. The relative values obtained by both methods showed good agreement. The ratio of the 30 kDa band to the 28 kDa band was higher for the alpha-actin-positive cells. The positive cells had ratios in the range of 0.33-0.82 (mean = 0.59), and six of the seven positive cell lines had a ratio greater than 0.5. The alpha-actin negative cell lines had ratios in the range of 0.21-0.43 (mean = 0.33). The difference in the TIMP-1 band ratio between the fibroblast and myofibroblast groups was significant (p = 0.013).

When the data were grouped by scleroderma cell lines and normal cell lines, the difference in the means was not as great (scleroderma mean = 0.533, normal mean = 0.433), and the difference was not significant. In this study a prominent 30 kDa TIMP-1 band appeared to be a marker for myofibroblasts in culture, independent of the source of the fibroblast cell line.

The results of the alpha-actin immunostaining and collagen and TIMP data are summarized in Table 4. For the most part, cell lines which were alpha-actin positive had high collagen and high TIMP levels and two major forms of TIMP-1. Conversely, the alpha-actin-negative cell lines were relatively low collagen and low TIMP producers and had less of a second TIMP-1 species in the media.




DISCUSSION

Our results demonstrate that the activated phenotype associated with scleroderma fibroblasts in culture is the myofibroblast phenotype. In our hands, cultured myofibroblasts expressed elevated levels of collagen, typical of scleroderma fibroblasts in culture (LeRoy, 1972; Buckingham et al., 1978; Uitto et al., 1979; Mayes et al., 1988).

For the most part myofibroblasts predominated from scleroderma biopsies whereas fibroblasts were the predominant cells grown from normal biopsies. Although not all scleroderma cell lines studied were high collagen producers, our data suggest that the scleroderma lines that were low collagen producers were of the normal fibroblast rather than the myofibroblast phenotype.

Scleroderma-derived cultures were not always myofibroblasts (alpha-actin positive), and cells derived from normal skin were not always normal fibroblasts (alpha-actin negative). In fact, normal fibroblasts predominated in scleroderma affected skin cultures in two cases and myofibroblasts predominated from a normal biopsy in one instance. These exceptions might be due to the nature of the biopsy or more likely due to variations in the stability of the fibroblast phenotype from different individuals. Additionally, subtle differences in the tissue culture environment may select for a certain fibroblast phenotype. Variable stability of fibroblast phenotypes was evident in this study, and this observation necessitated that all measurements obtained for a cell line be done at the same time.

Indirect evidence for the existence of myofibroblasts in scleroderma fibroblast cultures comes from collagen gel contraction studies by Gillery et al.(1991). On the whole scleroderma fibroblasts contracted the collagen gel more rapidly and more intensely than did normal cells. Lefebvre et al.(1992) have reported that myofibroblasts from granulation tissue contract collagen gels at a faster rate than do fibroblasts from the same source. These two studies combined imply a myofibroblast character to scleroderma cells. Evidence that myofibroblasts exist in scleroderma skin in vivo is provided by Sappino et al. (1990a) who reported positive smooth muscle alpha-actin immunostaining of biopsies of scleroderma skin.

In this study it was also found that myofibroblasts synthesize elevated levels of TIMP. The possible role of TIMP in the fibrosis of scleroderma has received little attention previously. TIMP protects collagen and other matrix macromolecules from degradation by metalloproteinases. Decreased collagenase activity has been found in scleroderma fibroblasts (Mayes and Kirk, 1991; Takeda et al., 1994) with normal levels of collagenase mRNA (Takeda et al., 1994). In scleroderma, high TIMP levels, combined with increased matrix synthesis, would exacerbate the fibrosis that is the hallmark of this disease. Additionally, TIMP-1 could be playing a role as a growth factor in scleroderma as its growth promoting activity has been shown in a number of cell lines in culture (Hayakawa et al., 1992).

The presence of two major forms of TIMP-1 in the media of cultured cells, as demonstrated by gel electrophoresis, appears to be characteristic of myofibroblasts. This doublet could be detected in all cultures but was more prominent in the alpha-actin-positive cell lines whether they were derived from normal skin or scleroderma affected skin. Clark et al.(1991) have separated out two forms of TIMP-1 in the media of WI38 fetal lung fibroblasts on the basis of concanavalin A binding, indicating differences in glycosylation. Clark et al. did not resolve the two species of TIMP-1 on reducing gels. Although we observed the best separations of the doublet on non-reducing gels, we also found evidence for two bands on reducing gels (data not shown).

Miyazaki et al.(1993) have separated multiple forms of TIMP-1 by isoelectric focusing. The six or more TIMP bands were presumably due to differences in the carbohydrate moieties. Differential glycosylation is also the most likely explanation for the two forms of TIMP-1 found in this present study. However, further study is needed to resolve the basis of the difference(s) in the two TIMP-1 forms.

Myofibroblasts are emerging as a significant cell type in tissue contraction and remodeling within numerous reactive lesions including cancer stroma, autoimmune fibrosing disorders, and wounds (for review, see Grinnell, 1994). The origin of myofibroblasts remains an area of debate. Myofibroblasts have been proposed to originate from fibroblasts, smooth muscle cells, or pericytes (Sappino et al., 1990b). Recent evidence supports the view that alveolar septal fibroblasts can be induced to modulate into typical myofibroblasts with bleomycin stimulation (Vyalov et al., 1993). Two recent studies by Desmoulière et al.(1993) and Ronnov-Jessen and Peterson(1993) show that TGF-beta1 can induce smooth muscle alpha-actin synthesis in cultured normal fibroblasts, indicating that TGF-beta plays an important role in myofibroblast differentiation. This activated phenotype then maintains elevated collagen production and TIMP levels after several passages in culture apparently without additional stimulation with TGF-beta.

TGF-beta2 and possibly other factors in inflammatory infiltrates may transform fibroblasts into myofibroblasts in the early stages of scleroderma (Kulozik et al., 1990). In terms of malignancies, cancer cells are a likely source of TGF-beta for myofibroblast differentiation in the tumor stroma (Ronnov-Jessen et al., 1992). Additionally, TGF-beta is known to increase collagen and TIMP synthesis in cultured fibroblasts (Roberts et al., 1986; Ignotz and Massague, 1986; Overall et al., 1989).

Our data provide additional evidence to support the belief that cultured fibroblasts are not a homogeneous population. This should be taken into account when evaluating data obtained from cultured fibroblasts. Discrepancies may result from the use of different phenotypic compositions of fibroblasts. In addition, the major phenotype seen in a fibroblast line may change with time in culture. Smooth muscle alpha-actin immunostaining provides an important characterization of the culture. We have observed differences in collagen and TIMP synthesis between normal fibroblasts and myofibroblasts. More differences are likely to emerge with further research.

Our experimental data further identify the scleroderma fibroblast in culture as a myofibroblast. The myofibroblast phenotype may provide a target for future therapeutic approaches in the treatment of scleroderma and other fibrotic diseases. Increasing knowledge of the myofibroblast and fibroblast differentiation will lead to a better understanding of scleroderma as well as all other reactive lesions where myofibroblasts are present.


FOOTNOTES

*
This work was supported in part by a grant from the Michigan Chapter of the Arthritis Foundation. 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: Div. of Rheumatology, Wayne State University, Hutzel Hospital, 4707 St. Antoine, Detroit, MI 48201. Tel.: 313-577-1133; Fax: 313-577-1938.

(^1)
The abbreviations used are: TIMP, tissue inhibitor of metalloproteinases; DMEM, Dulbecco's modified Eagle's medium; TGF, transforming growth factor.


ACKNOWLEDGEMENTS

We thank Dr. Henning Birkedal-Hansen for the gift of antibody to TIMP-1, Synergen for the gift of human TIMP-1, Charles Parzick for densitometric analysis of fluorographs, Dr. Jeffrey Moshier for gel radioactivity quantitation and critical review of the manuscript, Dr. William Stetler-Stevenson and Dr. Robert Karvonen for reviewing the manuscript and helpful discussion, and Dr. Shirley Russell for helpful discussion. We gratefully acknowledge the cooperation of normal and patient volunteers at the Scleroderma Research and Treatment Center, Hutzel Hospital Center for Rheumatic Diseases, Wayne State University.


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