(Received for publication, July 29, 1994; and in revised form, December 5, 1994)
From the
Cultured fibroblasts derived from skin biopsies from scleroderma
patients and normal individuals were examined for the presence of
smooth muscle -actin, a marker for myofibroblasts. Six of eight
scleroderma cell lines were found to be 50% or more positive for
-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
-actin
positive (myofibroblast enriched) cultures. In addition, the
myofibroblast-enriched cultures displayed a more prominent TIMP doublet
band pattern on SDS-polyacrylamide gel electrophoresis.
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 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 -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. ()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.
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.
Figure 1:
Panel A shows the typical staining
pattern observed for -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
-actin negative. A few actin fibers can be seen in some cells, but
the majority of cells do not show positive fluorescence. Magnification
is
125. 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 -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
-actin.
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 -actin-positive cell line derived from normal skin,
and one
-actin-positive scleroderma cell lines (S1). The cell line
from unaffected skin of a scleroderma patient and two affected
scleroderma cell lines (one
-actin positive and the other 50:50
-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 -actin-negative cell lines examined were relatively
low collagen producers. Four out of the six
-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 -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.
Figure 2:
Electrophoretic separation of TIMP-1
immunoprecipitates on 10 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 -actin-positive fibroblasts. Cell line N2 was
-actin negative and cell line N5 was not stained for
-actin.
Combining the TIMP and -actin data it was found that five of
the
-actin-positive cell lines had relatively high levels of TIMP
in the media while one had low TIMP. One
-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,
-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 -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
-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 -actin immunostaining and
collagen and TIMP data are summarized in Table 4. For the most
part, cell lines which were
-actin positive had high collagen and
high TIMP levels and two major forms of TIMP-1. Conversely, the
-actin-negative cell lines were relatively low collagen and low
TIMP producers and had less of a second TIMP-1 species in the media.
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 (-actin positive), and cells derived from
normal skin were not always normal fibroblasts (
-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 -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 -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-1 can induce smooth muscle
-actin synthesis in cultured
normal fibroblasts, indicating that TGF-
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-
.
TGF-2 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-
for
myofibroblast differentiation in the tumor stroma (Ronnov-Jessen et
al., 1992). Additionally, TGF-
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 -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.