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Heterogeneous synthetic phenotype of cloned scleroderma fibroblasts may be due to aberrant regulation in the synthesis of connective tissues.

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1221
HETEROGENEOUS SYNTHETIC PHENOTYPE OF
CLONED SCLERODERMA FIBROBLASTS MAY BE
DUE TO ABERRANT REGULATION IN THE
SYNTHESIS OF CONNECTIVE TISSUES
THERESA L. WHITESIDE, MARINA FERRARINI, PATRICIA HEBDA, and
ROBERT B. BUCKINGHAM
Clones of dermal fibroblasts from the skin of 4
normal subjects and 5 patients with progressive systemic
sclerosis (PSS; scleroderma) were established, and their
synthetic and proliferative characteristics were compared. A limiting-dilution assay was used to determine
frequencies of cloning in the microcultures of dermal
fibroblasts plated. The clones derived from single cells
were expanded in vitro and examined (in passages C-H)
for growth and synthesis of glycosaminoglycan (GAG)
and collagenase-sensitive protein (CSP). The clonogenicity of PSS fibroblasts was not significantly different
from that of normal fibroblasts. Normal fibroblast
clones were characterized by low levels of GAG and CSP
synthesis, and there was a correlation between the GAG
and CSP phenotypes. In contrast, clones of PSS fibroblasts were often, but not always, high producers of
GAG and CSP, but there was no correlation between the
Presented in part at the 49th Annual Meeting of The
American Rheumatism Association, Anaheim, CA, June 1985, and
at the Annual Meeting of the American Academy of Dermatology,
San Antonio, TX, December 1987.
From the Department of Pathology, Division of Clinical
Immunopathology, the Department of Dermatology, and the Department of Medicine, Division of Rheumatology and Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania.
Supported by NIH grant AM-24019, the Kroc Foundation,
the RGK Foundation, Austin, Texas, and the Western Pennsylvania
Chapter of the Arthritis Foundation.
Theresa L. Whiteside, PhD: Associate Professor, Department of Pathology; Marina Ferrarini, MD: Postdoctoral Fellow,
Department of Pathology; Patricia Hebda, PhD: Assistant Research
Professor, Department of Dermatology; Robert B. Buckingham,
MD: Clinical Associate Professor, Department of Medicine.
Address reprint requests to Theresa L. Whiteside, PhD,
Room 5725, One Children’s Place, 3705 Fifth Avenue at DeSoto
Street, Pittsburgh, PA 15213-3417.
Submitted for publication December 8, 1987; accepted in
revised form April 25, 1988.
Arthritis and Rheumatism, V d . 31, No. 10 (October 1988)
levels of GAG and CSP synthesis. It appears that
scleroderma skin is composed of fibroblast clones that
are unable to regulate the synthesis of connective tissue
components and often synthesize large amounts of connective tissue macromolecules.
Progressive systemic sclerosis (PSS; scleroderma) is a disease of unknown cause and is characterized by fibrosis and excessive accumulation of
connective tissue in the skin and internal organs (1-4).
Pathogenetic mechanisms that may be responsible for
this fibrosis include “activation” and “selection” of
connective tissue fibroblasts (5,6). Activation by an
unknown stimulus could convert the cell from a normal producer of collagen and glycosaminoglycan
(GAG) to a high producer of collagen and GAG.
Fibroblast selection, on the other hand, implies that
within a heterogeneous population of normal fibroblasts, high producers of connective tissue are favored, by unknown processes, to replace cells that are
low producers. Dermal fibroblasts obtained from patients with PSS and grown in vitro as mass cultures
synthesize more connective tissue, have higher levels
of messenger RNA for collagen, and exhibit different
growth characteristics compared with normal dermal
fibroblasts (5,7-12). In addition, the affected skin of
scleroderma patients is thickened and contains excessive amounts of connective tissue (2-4).
Human dermal fibroblasts cultured in vitro represent mixtures of fibroblasts with various synthetic
and proliferative capabilities. Because the behavior of
mass cultures is determined by cell-cell interactions, it
is necessary to separate and clone individual fibroblasts if their growth and synthetic capacities are to be
evaluated. Cloning of dermal fibroblasts allows for the
WHITESIDE ET AL
1222
functional analysis of normal or diseased skin at the
level of a single cell. After clonal expansion, individual
clones can b e characterized independently of the in-
fluences and modulators that operate in mass cultures.
Furthermore, once established in vitro, clones of
normal fibroblasts can be manipulated and assessed
for the effects of external factors, such as serum or
cytokines, on their function.
We cloned dermal fibroblasts from cell lines
established from skin biopsy material from normal
adults and from adult patients with PSS, a n d we
compared their growth rates a n d the biosynthetic
phenotype.
MATERIALS AND METHODS
Mass cultures of dermal fibroblasts. Full-thickness
7-mm punch biopsy specimens were obtained from the
dorsal forearm, midway between the wrist and the elbow, of
normal adult volunteers and from adult patients with early,
rapidly advancing PSS. The control group was matched for
sex and age with the patient group. At the time of biopsy,
none of the patients were receiving medications known to
affect connective tissue metabolism. Specifically, no patient
was taking corticosteroids, D-penicillamine, or cytotoxic or
immunosuppressive agents. All patients were classified as
having PSS with diffuse scleroderma, according to previously published criteria (13). All had active, rapidly progressing disease at the time of the skin biopsies, and all PSS
biopsy samples were taken from involved skin.
Fibroblast cultures were established from the explants (10 normal subjects and 10 patients with PSS) through
subculture of cells outgrowing from the lowest portion of the
dermis and serial passage, as described previously (8).
Cultures were grown in CMRL 1066 medium with Earle’s
salts (Gibco, Grand Island, NY) supplemented with 15%
pooled human serum (obtained from donors at our institution), 100 unitslml of penicillin, 100 pg/ml of streptomycin, 2
mM L-glutamine, 2 mM L-proline, and ascorbic acid (0.5 pg/
well). Cultures in the fourth or fifth passages were cloned.
Fibroblast cloning. Confluent cultures of normal or
PSS fibroblasts were trypsinized using a 0.25% solution of
trypsin (Gibco), washed in medium, checked for viability
with trypan blue dye, and diluted in growth medium containing 50% (volumeivolume) pooled fibroblast-conditioned medium (FCM; the supernatant from confluent normal dermal
fibroblast cultures). The limiting-dilution assay was carried
out, and fibroblasts were plated in 96-well flat-bottom plates
(Falcon, Oxnard, CA) at concentrations of 2, 1 , and 0.5
fibroblasts per well. One 96-well plate was used for each cell
concentration. The plates were incubated at 37°C in an
atmosphere of 5% CO, in air. They were examined daily
with an inverted-phase microscope and were scored for
growth during the next 3 weeks. Estimates of the minimum
number of proliferating fibroblasts were obtained by the
minimum chi-square method from the Poisson distribution
relationship between the number of cells and the logarithm
of the percentage of nonresponding (negative) microcultures, as described by Taswell (14).
Only the plates seeded with 0.5 fibroblasts per well
were used in the selection of clones for expansion. ‘These
plates were observed daily, and microcultures originating
from single cells were identified. These microcultures were
transferred to 24-well plates (Falcon) upon reaching confluence (2-3 weeks). The clones were expanded in these 24-well
plates, transferred to 25-cm2 Falcon flasks, and further
expanded in medium containing FCM. Clones were either
passaged serially (every 7-8 days) or cryopreserved in 10%
DMSO plus 20% pooled human serum.
Assays of fibroblast clones. Clones were grown in
1099 medium without FCM before being assayed for GAG
and collagenase-sensitive protein (CSP) synthesis. Triplicate
wells were inoculated with 2 x lo4 cloned fibroblasts. On
day 2, wells were fed with complete growth medium, and on
day 4, wells were fed with glucosamine-free or proline-free
medium. On days 4 and 6, cultures were pulsed with 4 pCi/
ml of D-( 1,6-3H-N)-glucosamine hydrochloride (specific activity 45 Ci/mmole) or with 2 pCi/ml of L-(S3H-proline)
(specific activity 12.9 Cdmmole; both from New England
Nuclear, Boston, MA) and were harvested 12 hours later.
All clones were studied in passages C and D. Selected
clones, which grew well as microcultures, were also studied
for in vitro connective tissue synthesis in later passages
(from E to H).
Determinations of GAG and CSP synthesis in fibroblast microcultures. After detaching the fibroblasts from the
wells, as described previously (15), the cell numbers were
counted in a Coulter counter (Coulter, Hialeah, FL). These
numbers were used to calculate the levels of GAG and CSP
synthesis per cell. In each of the triplicate wells, GAG
synthesis was estimated as the counts per minute of ’Hglucosamine incorporated into total GAG (cells + culture
medium) and precipitable by cetylpyridinium chloride, as
described previously (15). The CSP synthesis was determined in triplicate wells as the cpm of 3H-proline incorporated into total acid-soluble, collagenase-sensitive material,
using the method of Peterkofsky and Diegelman (16), with
our modifications as described elsewhere (17).
Statistical analyses. Linear regression analysis was
used to examine the relationship of the in vitro levels of
connective tissue synthesis by normal fibroblast clones to
the levels of synthesis by PSS fibroblast clones. Student’s
t-test was used to determine the significance of these correlations and differences in cloning efficiency and differences
in cell numbers.
RESULTS
Cloning of normal a n d PSS fibroblasts. PSS and
normal dermal fibroblasts growing as short-term cultures were trypsinized a n d cloned by the limitingdilution method. The cloning frequencies of 4 normal
and 4 PSS cultures were determined using Taswell’s
statistical program (14) (Figure 1). Both the normal
a n d the PSS fibroblasts cloned well in this system, and
although normal fibroblasts h a d a somewhat better
CLONING OF PSS FIBROBLASTS
1223
PSS DERMAL FIBROBLASTS
FIBROBLASTS / WELL
j
--I
CLONING EFFICIENCY:
' 5t
1
10
(95% CONFIDENCE LIMITS)
A 0.30(0.20-0.38)
E 0.35(0.24-0.45)
m 0.53(0.4I -0.65i
D 0.60(0.46-0.74)
NORMAL DERMAL FIBROBLASTS
FIBROBLASTS /WELL
0.5
I
I.5
I
9
2
2.5
I
I
68-
-I
w
'
w
46-
2
8
'
32-
z
W
0
22 -
f
CLONING EFFICIENCY:
W
(35% CONFIDENCE LIMITS)
IX
15-
I
L
10
W
D
T
H
0.58(0.43-0.73)
0.64(0.48-0.80)
0.78(0.60-0.95)
0.67(0.54-0.80)
D
T
T
\
'
Figure 1. Cloning frequencies of dermal fibroblasts obtained from
patients with progressive systemic sclerosis (PSS) and from normal
subjects, determined by the method of Taswell (14). The letters
indicate the different strains of dermal fibroblasts.
clonogenicity than PSS fibroblasts, the difference was
not statistically significant. These experiments were
performed first to establish the validity of our approach. After that, we cloned at least 10 PSS and 10
normal fibroblast lines. The mean cloning efficiency of
PSS fibroblasts was not significantly different from
that of normal fibroblasts (0.57 versus 0.62). If it had
been significantly different, the comparisons of the
biosynthetic properties would not have been valid.
We established, expanded, and characterized
36 normal fibroblast clones and 37 PSS fibroblast
clones. The normal clones were derived from the skin
of 4 normal volunteers (strains MIT, LIN, DOL, and
DiMo) who were sex- and age-matched with the patients; the PSS clones were derived from the dermis of
5 patients with PSS (strains CD, HB, OA, HU, and
JAD). The normal and PSS parent strains were established from the lower portion of the dermal biopsy
samples and cloned in pairs (normal with PSS: MIT
with CD, LIN with HB, DOL with OA, DOL with
HU, and DiMo with JAD). Only the clones that were
derived from single cells and were strong growers
were selected for further expansion. Some of these
selected clones were studied at an early passage (C or
D) and then again at later passages (E through H).
Given the plating concentration used (0.5 cells/well)
and the stringent microscopy of each well after plating,
it is likely that our microcultures were clonally derived, and operationally, we refer to them as clones.
Growth of normal and PSS clones. Counts of
viable cells were obtained on all clones at the time of
the CSP and GAG determinations (Figure 2). On day
4, the cell counts for 36 normal clones ranged from
45,00O/well to 180,00O/well, and the mean cell count
(? 1 SD) was 88,000 ? 34,00O/well. Cell counts for 37
PSS clones ranged from 30,000 to 115,00O/well, and
the mean (& 1 SD) cell count was 64,000 k 22,00O/well.
Although PSS fibroblast clones did not grow as well as
normal fibroblast clones under the in vitro conditions
used, the difference in cell numbers was not statistically significant. In mass cultures, PSS and normal
fibroblasts had similar growth kinetics, even though
PSS cultures often reached confluence at a lower total
cell number (data, based on a comparison of 10 normal
and 10 PSS strains, not shown). This has been the case
in all growth experiments on PSS and normal cells in
our laboratory, beginning with our early studies (8). It
is interesting that the proliferation of the parent
strains, both normal and PSS, was better than that of
the individual clones in all cases studied (Figure 2).
Cell counts were also determined for mass
cultures and clones on day 6 of proliferation. These
were comparable with the day 4 counts (data not
shown), and it can be concluded that most lines and
clones achieved the stationary phase of growth by day
4 under the in vitro conditions used.
Biosynthetic characteristics of normal and PSS
clones. GAG and CSP syntheses were compared in
normal and PSS clones. As shown in Figure 2, PSS
clones synthesized more GAG per cell than did normal
clones (P < 0.001) on day 4 of culture. Comparable
WHITESIDE ET AL
1224
PSS FIBROBLAST CLONES
A
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NORMAL FIBROBLAST CLONES
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700T
M IT
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3
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9
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600
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180 x
160
d
.
400
a-;200
300
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120
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2
;
80
f
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A
20
PI1
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5
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7
8
9
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Figure 2. Glycosaminoglycan (GAG) synthesis (m)and numbers of cells (a)
in A, progressive systemic sclerosis (PSS)
clones (n = 37) and B, normal dermal fibroblast clones (n = 36) on day 4 of growth (medium changes on days 2 and 4;see
Materials and Methods for details). Overall, the PSS clones produced significantly more GAG in vitro than did normal
fibroblast clones (P< 0.001). Letters and numbers across the bottom and letters at the top indicate the biopsy specimen
from which the clones were obtained. Values are the mean
1 SD of triplicate wells.
*
results were obtained for the d a y 4 cultures (data not
shown). The numbers of cells for each clone at the
time of GAG and CSP assays are shown in Figure 2.
We have previously shown that in actively growing
mass cultures of normal dermal fibroblasts, GAG
synthesis was inversely related to cell density (18). We
also found that the level of GAG synthesis per cell was
higher in early log-phase cultures than in the confluent
or stationary-phase cultures (18). In the present study,
there was no clear-cut, one-to-one relationship between cell density and GAG synthesis in normal or
PSS fibroblast clones (Figure 2 ) . Nevertheless, there
were higher numbers of cells in 4-day cultures of
normal clones, which as indicated above, synthesized
less GAG than did the PSS clones.
The PSS clones derived from a single parent
line were heterogeneous in GAG synthesis, and some
clones produced high levels of GAG per cell, even
when their relative cell numbers were not low. Clones
derived from PSS patient HU were an exception. They
did not show this heterogeneity, were all relatively low
GAG producers, and proliferated better than normal
fibroblast clones (DOL) that were grown and assayed
simultaneously.
The synthesis of CSP by normal fibroblast
clones was generally low on day 4, except for those
CLONING OF PSS FIBROBLASTS
1225
< 0.05) levels of CSP synthesis and more heterogeneity in CSP production than did those derived from
normal fibroblast lines (Figure 3 ) . Most of the clones
from the PSS parent line CD, which had a relatively
derived from the MIT parent line (Figure 3B). This line
showed higher CSP production in vitro than did the
other normal lines we studied. Overall, the clones
derived from PSS lines showed significantly higher ( P
PSS FIBROBLAST CLONES
A
OA
HB
810
v)
IB
0
<z
632
I
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LL
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I
I
I
I
I
I
I
I
I
300
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I
I
I
I
I
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I
I
I
I
I
I
I
I
I
!J
d A 2
JAD
HU
50
PN
I
I
I
%E884A2H7n
PA?JnG7G4F2
PA4 F O DI D 3
C4 FII 812
1
NORMAL FIBROBLAST CLONES
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
LIN
I
I
I
I
I
II
II
II
DOL
II
1
I
I
I
I
I
I
I
I
I
I
I
I
I
I1
II
II
I1
I
I
I
I
I
I
II
I
II
Dilllo
I
I
I
X
200
I
I
I
4b
P
g
100
Figure 3. Synthesis of collagenase-sensitive protein (CSP) by A, progressive systemic sclerosis (PSS) and B, normal
dermal fibroblast clones o n day 4 of growth. The difference in CSP synthesis by PSS fibroblast clones and by normal
fibroblast clones was significant (P< 0.05). See Figure 2 for other definitions.
WHITESIDE ET AL
1226
0
NORMAL FW?O&AST CLONES ( n - 6 )
FIBROBLAST CLONES (n=15)
A PSS
I
1100
1000
900
800
:
d
[z
700
600 b
\
500
400
-
2
300 2
200
5
I00
CSP ( cpm x
10" FIBROBLASTS )
Figure 4. The relationship between glycosaminoglycan (GAG) and
collagenase-sensitive protein (CSP) synthesis in normal (subject
MIT) and progressive systemic sclerosis (PSS) (patient CD) fibroblast clones on day 4 of growth. The data are representative of all
other pairs of normal and PSS fibroblast clones studied and analyzed
in the same way.
high level of CSP synthesis in mass culture, had a
higher synthetic phenotype than that of other PSS
clones.
Figure 4 illustrates the relationship between
CSP and GAG synthesis by normal and PSS fibroblast
clones on day 4 of culture. All of the normal clones we
studied were characterized by uniformly lower synthetic potential for both GAG and CSP. For example,
14 of the 16 normal MIT clones (paired with PSS CD
clones) (Figure 4) had a low synthetic phenotype and
showed good correlation between the levels of GAG
and CSP synthesized (r = 0.856). Linear regression
analysis performed with 36 normal fibroblast clones
for GAG synthesis versus CSP synthesis gave a correlation coefficient of 0.558, indicating that normal
dermal fibroblasts were consistently low producers of
both GAG and CSP, and showed a good correlation
between GAG and CSP production.
In contrast, 13 of 15 PSS clones (Figure 4) were
high producers of connective tissue components, but
no relationship between the levels of GAG and CSP
synthesized by these clones could be established.
Most PSS clones that were high producers of either
GAG or CSP (26 of 37) manifested this aberrant
regulation of connective tissue synthesis as a lack of
correlation between GAG and CSP phenotypes. Some
PSS clones (1 I of 37), however, behaved in a manner
similar to the normal clones, i.e., exhibited low synthetic phenotype and a good correlation between GAG
and CSP production. For example, all 6 clones derived
from the skin of patient HU were low producers of
both GAG and CSP. Patient HW had a very active,
rapidly progressive disease of 47 months duration at
the time the skin biopsy was obtained.
Stability of the synthetic phenotype in fibroblast
clones. Several of the normal and PSS fibroblast clones
were tested for GAG and CSP synthesis at 2 or 3
different passages in vitro (C through H). As shown in
Table 1, the normal fibroblast clones tended to show
increases in levels of GAG synthesis per cell between
passages C and E. In contrast, PSS fibroblast clones
tended to decrease their GAG synthesis between passages C and E, F, G, or H. The reason for this is not
clear. Normal clones grew substantially better than
PSS clones in later passages in vitro, and the differences in synthesis may be related to the growth
patterns of these clones.
The levels of CSP synthesis remained basically
unchanged in different passages for both normal and
PSS fibroblast clones. It appears that the CSP phenotype of dermal fibroblast clones is a more stable
characteristic in culture than is the GAG phenotype.
DISCUSSION
Clonal analysis performed with normal and PSS
adult dermal fibroblasts indicates that scleroderma
skin is composed of cells that are heterogeneous in the
synthesis of GAG and CSP, show no correlation
between the amounts of GAG and of CSP produced in
vitro, and often, but not always, produce elevated
levels of connective tissue components in vitro. PSS
clones were relatively uniform in their expression of
the high synthetic phenotype of GAG, but showed
wide variability in the synthesis and accumulation of
CSP. In contrast, normal skin appeared to contain
fibroblasts that were low producers of GAG and CSP
when cloned in vitro. These conclusions, however, are
based on a limited number of in vitro experiments with
73 clones of adult dermal fibroblasts. These clones
represent a small number of those that were obtained.
The requirements for expansion and maintenance of
clones are rigorous, and it was not possible to test
most of them. Having selected clones for expansion,
1227
CLONING O F PSS FIBROBLASTS
Table 1. Stability of the GAG and CSP phenotypes of selected
normal and PSS dermal fibroblast clones studied in different passages*
Clone, passage
GAG
CSP
Normal clones
MIT 5
C
E
MIT 7
C
497 t 7
697 2 20
500 t SO
125 t 10
124 +- 4
146 t 71
515 t 30
160 t 20
187 t 40
155 t 9
240 t 20
340 t 20
135 t 20
135 t 20
240 t 6
388 t 20
130 t 30
90 t 3
78 t 30
395 t 50
133 t 20
148 t 5
67 t 30
321 t 40
91 t 8
120 t 8
46 t 10
362 2 30
141 t 10
127 t 10
160 t 4
130 t 25
50 t 20
50 t 20
200 t 20
135 t 10
120 t 40
105 10
160 2 20
80 t 10
140 t 30
85 ? 25
120 t 10
275 t 40
60
155
500 t 40
397 t 45
240 t 60
80 2 10
275 t 45
245 t 20
220 t 30
136 t 10
610 t 40
361 & 30
80 t 10
80 t 15
D
E
MIT 9
C
E
MIT
C
E
MIT
D
E
MIT
D
E
MIT
D
E
11
13
14
16
PSS clones
HU C4
C
H
HU F11
D
H
HU DI
C
H
HU B12
D
G
OA G4
C
F
OA G7
C
E
OA F2
C
E
*
2
2
7
30
* Expanded clones were passaged as described in Materials and
Methods. Cells were inoculated into wells (2 x 104/well) and
supplied with fresh medium every 2 days. On day 4, triplicate
cultures were pulsed for 12 hours, harvested, and assayed for
glycosaminoglycan (GAG) and collagenase-sensitive protein (CSP).
Values are the mean 2 SD of 3 determinations, and are expressed as
cpm x 10-3/106fibroblasts.
we were not able to repeatedly passage many of them
in vitro. Thus, the 36 normal and 37 PSS fibroblast
clones (obtained from 4 normal and 5 PSS skin biopsy
samples) we studied were those that were best able to
survive and proliferate under the in vitro culture
conditions we established. We do not know whether
these clones are sufficiently representative of a full
clonal repertoire of the skin. However, the demanding
nature of these experiments precludes clonal expansion and analysis of every clone outgrowing from a
parent line.
The comparably high cloning efficiency of normal and PSS fibroblast cultures indicated that both
were equally capable of cloning in our system. Our
cloning efficiency was comparable with that reported
by Korn et a1 (19) for foreskin fibroblasts and with that
reported by Brinckerhoff and Nagel (20) for rabbit
synovial fibroblasts. Still, only 3040% of the cells
plated gave rise to microcultures for different fibroblast strains, and a possibility remains that microcultures generated may not accurately represent the
complete cellular content of the parent populations
cloned.
It is remarkable that the 2 groups of randomly
selected normal and PSS fibroblast clones exhibited
such clearly different patterns of connective tissue
synthesis. The normal fibroblast clones were characterized by the low synthetic phenotype and were
relatively homogeneous in its expression, producing
low amounts of both GAG and CSP in culture. In
contrast, the PSS clones included a mixture of high
and low producers of connective tissue components.
Differences in growth of normal and PSS clones were
not statistically significant, and proliferation of both
types of clones was lower than that of their parent
lines (Figures 2 and 3). This probably indicates that for
optimal proliferation and cloning of dermal fibroblasts,
soluble products of other cells and conditioned medium are required.
The relationship between cell growth and synthesis of connective tissue components is not straightforward in fibroblast clones. While mass fibroblast
cultures in the log phase of growth exhibited an
inverse relationship between the number of cells and
the accumulation of GAG in our earlier studies (18),
such was not the case for fibroblast clones in this study
(see Figure 2 ) . Also, several normal clones that were
reassessed for GAG synthesis and cell counts at later
passages showed higher cell counts and increased
GAG synthesis compared with those determined at an
earlier passage. In contrast, CSP synthesis remained
relatively unchanged in the normal clones tested at
several passages. The issue of growth versus CSP and
GAG synthesis is important because it has been sug-
WHITESIDE ET AL
1228
gested that the primary lesion of fibrosis in scleroderma is related to the regulation of cell growth rather
than connective tissue synthesis (21). The results of
our experiments with fibroblast clones do not support
this hypothesis. The lines and clones of PSS fibroblasts did not show impressive growth differences
compared with normal clones/lines, and the differences that were identified could not be directly related
to the connective tissue biosynthesis.
The one plausible explanation for the observed
differences between normal and PSS clones may be
related to the regulation of connective tissue biosynthesis in the cell. Clearly, no relationship could be
established between CSP and GAG synthesis in PSS
clones. In contrast, such a relationship did exist in
normal clones. This finding suggests that PSS fibroblast clones with the high synthetic phenotype had a
dysregulation in the mechanism necessary for orderly
deposition of the connective tissue matrix. Those PSS
clones (obtained from skin biopsy samples from patients OA and HU) that produced low levels of CSP
and GAG in vitro appeared to be regulated normally. It
is not clear why clones and lines derived from 2
patients with PSS did not express a high CSP phenotype. We could identify no clinical differences among
the 5 patients that would explain this phenomenon.
It has been difficult to identify the mechanism of
fibroblast activation in PSS. Our data do not support
activation as the pathogenetic mechanism in PSS:
Only some, not all, of the PSS clones were high
producers of GAG and collagen. Neither did we obtain
evidence for the selection hypothesis: The dysregulated phenotypes that synthesized high levels of one
connective tissue component but not the other were
found only among the PSS clones, never among the
normal clones. At the clonal level, a heterogeneous
expression of a biosynthetic phenotype by PSS, but
not normal, fibroblast clones suggests that more than
one factor may be responsible. Although most of the
PSS clones expressed a high synthetic phenotype that
was stable in different passages, we remain ignorant as
to the event(s) responsible for the derivation of this
phenotype. It is possible that a combination of selection and activation events could account for its appearance. A variety of mechanisms, including serum factors (6,22) and products of mononuclear cells (15,17,
23), have been proposed as effectors of fibrotic
changes in PSS and other diseases.
The ability to clone and maintain in culture
normal adult and fetal dermal fibroblasts should be
useful in future in vitro studies to identify factors that
are capable of changing the growth and/or the phenotype of adult fibroblasts.
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1. Rodnan GP: Progressive systemic sclerosis (scleroderma), Arthritis and Allied Conditions. Ninth edition.
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1971
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scleroderma and scleredema. J Invest Dermatol58:129132, 1979
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Arthritis Rheum 25:189-195, 1982
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systemic sclerosis (PSS, scleroderma) dermal fibroblasts
synthesize increased amounts of glycosaminoglycan. J
Lab Clin Med 101:659-669, 1983
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Increased collagen accumulation in dermal fibroblast
cultures from patients with progressive systemic sclerosis (scleroderma). J Lab Clin Med 925-21, 1978
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902-903, I977
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messenger RNA steady-state levels by retinoids. Arthritis Rheum 30:404-411, 1987
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CLONING OF PSS FIBROBLASTS
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Peterkofsky R, Diegelman R: Use of a mixture of
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Whiteside TL, Buckingham RB, Prince RK, Rodnan
GP: Products of activated mononuclear cells modulate
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1229
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phenotype in normal fibroblasts after prolonged exposure to soluble mediators from mononuclear cells.
Arthritis Rheum 2954-64, 1986
Korn JH, Torres D, Downie E: Clonal heterogeneity in
the fibroblast response to mononuclear cell derived
mediators. Arthritis Rheum 27: 174-179, 1984
Brinckerhoff CE, Nagel JE: Collagenase production by
cloned populations of rabbit synovial fibroblasts. Coil
Relat Res 5:433-444, 1981
LeRoy EC, Mercurio S, Sherer GK: Replication and
phenotypic expression of control and scleroderma fibroblasts: response to growth factors. Proc Natl Acad Sci
USA 79: 12861290, 1982
Bordin S, Page RC, Narayanan AS: Heterogeneity of
normal human diploid fibroblasts: isolation and characterization of one phenotype. Science 223:171-173, 1984
Korn JG: Fibroblast PGE, synthesis: persistence of an
abnormal phenotype after short term exposure to mononuclear cell products. J Clin Invest 71:1240-1246, 1983
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aberrant, due, regulation, tissue, synthetic, synthesis, may, connection, heterogeneous, phenotypic, scleroderma, clones, fibroblasts
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