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Inhibition of excessive scleroderma fibroblast collagen production by recombinant ╨Ю╤Ц-interferonAssociation with a coordinate decrease in types I and III procollagen messenger RNA levels.

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85 1
INHIBITION OF EXCESSIVE SCLERODERMA
FIBROBLAST COLLAGEN PRODUCTION BY
RECOMBINANT ?.-INTERFERON
Association with a Coordinate Decrease in Types I and I11 Procollagen
Messenger RNA Levels
JOEL ROSENBLOOM, GEORGE FELDMAN, BRUCE FREUNDLICH, and SERGIO A. JIMENEZ
The effects of recombinant y-interferon (rec yIFN) on collagen production by confluent monolayer
cultures of progressive systemic sclerosis (PSS) dermal
fibroblasts were studied. Five cell lines obtained from
patients with rapidly progressive disease of recent onset
were examined. All PSS fibroblast cell lines exhibited
increiased collagen production when compared with
normal skin cell lines. It was found that rec y-IFN
caused potent inhibition of PSS fibroblast collagen production in a concentration-dependent manner. Greater
than 50% inhibition was observed with as little as 50
antiviral unitslml, and maximal effects were attained at
a concentration of 500 unitslml. The rec y-IFN caused
reprsducible inhibition of collagen production by the 5
PSS fibroblast cell lines, ranging from 58.9% to 85.6%
of control values. Measurement of type I and type I11
procaillagen messenger RNA (mRNA) levels with specific
complementary DNA probes demonstrated a coordinate
reduction of >60% in mRNA for both transcripts in rec
y-IFN-treated cells, compared with control cells. These
findings indicate that rec y-IFN can modulate the excessive collagen biosynthesis characteristic of PSS fibro-
Presented in part at the 49th Annual Meeting of the American Rheumatism Association, Anaheim, CA, June 1985.
From the Collagen Research Laboratories, Rheumatology
Section, Department of Medicine, School of Medicine and the
Center for Oral Health Research, School of Dental Medicine,
University of Pennsylvania, Philadelphia.
Supported by NIH grant no. AM-19616.
Joel Rosenbloom, MD, PhD; George Feldman, PhD; Bruce
Freuncllich, MD; Sergio A. Jimenez, MD.
Address reprint requests to Sergio A. Jirnenez, MD, Room
570, Mlaloney Bldg., Hospital of the University of Pennsylvania,
3600 Spruce Street, Philadelphia, PA 19104.
Submitted for publication November 20, 1985; accepted in
revised form January 9, 1986.
Arthritis and Rheumatism, Vol. 29, No. 7 (July 1986)
blasts and that this effect can be explained largely by the
y-IFN-mediated decrease in specific collagen mRNAs.
Scleroderma (progressive systemic sclerosis,
PSS) is a connective tissue disease of unknown etiology, characterized by the deposition of excessive
amounts of collagen and other connective tissue macroromolecules in the skin and various internal organs
(1). Although the pathogenesis and mechanisms responsible for the excessive tissue collagen accumulation in PSS are not known, it is apparent that some of
the most significant clinical manifestations of the disease result from tissue damage caused by the progressive fibrotic process. At present, there is no effective
treatment for PSS, and frequently, the relentless progression of fibrosis causes severe functional and structural alterations in the affected organs and eventual
death.
We have recently discovered that y-interferon
(y-IFN) is a potent and selective inhibitor of collagen
production by normal human dermal fibroblasts. Addition of as little as 500 antiviral unitdm1 of y I F N to
confluent cultures of these cells resulted in >80%
inhibition of collagen production, with minimal effects
on cell viability or on total protein synthesis (2).
Subsequently, we demonstrated that inhibition of fibroblast collagen synthesis was a constitutive property
of y-IFN since these effects were reproduced with
recombinant y-interferon (rec y-IFN) (3). The modulation of fibroblast collagen synthesis by rec y-IFN
probably occurred at a transcriptional level since the
IFN-treated fibroblasts displayed a concomitant reduction in levels of type I procollagen messenger RNA
(mRNA) (3). These results have recently been confirmed by other investigators (43).
852
ROSENBLOOM ET AL
''C-hydroxyproline production by cultured dermal fibroblasts from progressive systemic
sclerosis (PSS) patients and from age- and sex-matched controls*
Table 1.
Normal fibroblasts
Cell line
Agelsex of
subject
PSS fibroblasts
''C-hydroxyproline
(dpm x 10-3/dish)
Cell line
Agelsex of
patient
''C-hydroxyproline
(dpm x 10-3/dish)
N- 1
46lM
9.23
9.12
8.37
7.96
KA
43lM
12.65
12.97
11.51
11.38
N-2
28lF
9.41
8.39
8.15
so
28lF
33.48
29.07
27.50
N-4
62iF
9.75
GR
65iF
21.01
18.91
KM
38lF
10.60
PR
44/F
14.90
N-44
28iF
11.20
SB
28lF
26.72
Mean t SD
9.22
* 1.07t
20.01
2
6.45t
* Fibroblasts from patients and controls were cultured under identical conditions. After confluency,
they were labeled with 14C-prolinein the presence of ascorbate and Paminopropnonitrile. After
labeling, the media were removed and extensively dialyzed. Aliquots were then hydrolyzed and assayed
for their ''C-hydroxyproline content by a specific chemical assay (10). See Patients and Methods for
details. Each value represents the average of results from separate experiments which were performed
in either duplicate or triplicate.
tP < 0.0016, by Student's r-test
The purpose of the present study was to determine if rec y I F N could modulate the excessive
collagen biosynthesis that is characteristic of PSS
fibroblasts. We found that rec yIFN is a potent
inhibitor of collagen production by PSS fibroblasts and
that this effect appears to be mediated largely by a
coordinate decrease in types I and I11 procollagen
mRN.A levels.
PATIENTS AND METHODS
Source of rec yIFN. Preparations of rec y-IFN were
kindly supplied by Dr. Sidney Petska (Hoffman-LaRoche,
Nudey, NJ) and were standardized to contain 1,000 unitshl
of antiviral activity, based on the cytopathic inhibition assay
described by Rubinstein et al (6).
PSS fibroblast cell lines. PSS dermal fibroblast cell
lines were obtained from 5 patients who fulfilled the American R.heumatism Association criteria for the diagnosis of
PSS (7'). Only patients who had active and rapidly progressive disease and who had not received any treatment known
to influence connective tissue metabolism were studied. All
5 patients had clinical evidence of internal organ involvement. In all cases, the duration from the onset of symptoms
to the time of the study was < 1 year.
Full-thickness skin biopsy specimens were obtained
from the leading edge of affected skin from untreated patients, with their informed consent. Monolayer cultures of
dermal fibroblasts were established from these tissues, as
described previously (8). All aspects of the research were
carried out according to the guidelines of the Declaration of
Helsinki.
Fibroblast cultures and labeling conditions. The PSS
dermal fibroblasts obtained as described above were grown
in Eagle's minimum essential medium supplemented with
10% fetal calf serum (FCS) in a 5% COz atmosphere. The
cells (between passages 6 and 14) were plated at a density of
19,000/cm2in 30-mm plastic culture dishes and were cultured
for 6 days to allow the cultures to reach confluency before
initiation of these experiments. Previous studies demonstrated that under these conditions, the cultures were contact-inhibited, since they did not display detectable cell
division as measured by serial cell counts, DNA assays, or
3H-thymidine incorporation. To avoid any influence of the
rec y I F N on fibroblast proliferation, only confluent cultures
were used (9).
On day 7, the cultures were inspected with a phasecontrast microscope to assure their confluency. The media
were then removed, and 1.5 ml of fresh media containing
various dilutions of rec y I F N , 5% FCS, and 50 pdml
ascorbate was added. In certain experiments, 10% FCS was
used, but because the results were essentially identical, the
lower serum concentration was used for all subsequent
studies. After the cultures were exposed for 2 days, the
media were again replaced with the same media containing,
in addition to rec y-IFN, 4 pCilml of ''C-proline and 100
pg/ml of Paminoproprionitrile, and the incubation was
continued for 24 hours. Control cultures were handled
IFW ]MODULATION OF PSS FIBROBLAST COLLAGEN PRODUCTION
exactly as described above, except that the culture media did
not contain rec yIFN.
*4C-hydroxyproline assay. The amount of I4Chydroxyproline, representing the collagen produced by the
control and rec y-IFN-treated fibroblasts at the end of the
24-hour incubation with 14C-proline, was measured by a
specific chemical assay (10). Only the culture media were
assayed since, as shown previously, >85% of total collagen
produced by the control or experimental cultures is released
into tbe media under the conditions employed (11).
Isolation and analysis of total RNA from untreated and
rec y-IFN-treated scleroderma skin fibroblasts. For isolation
and analysis of total RNA, the PSS fibroblasts which produced the greatest amount of collagen (line SO) were cultured to confluency as described above, but in T175 plastic
flasks, and then the medium was replaced with fresh medium
that contained 50 p g h l ascorbic acid and 10% FCS. After 24
hours, the medium was again replaced with fresh medium
containing 50 pghl ascorbic acid, with or without 500
units/nil of rec yIFN.
After 48 hours, the medium of 2 T175 flasks of either
treated or untreated cells (corresponding to 8-10 x lo6 cells)
was decanted, and the cells were washed twice with 5.0 ml of
0.02% versene. The cells were then scraped off the flasks and
suspended in 10 ml of 0.02% versene. The cell suspensions
were combined and then centrifuged at 600g for 5 minutes.
The cell pellets were then resuspended by gently vortexing
in 5 volumes of 4M guanidinium isothiocyanate, 5 mM
sodiurn citrate, 0.1M P-mercaptoethanol, and 0.5% sarkosyl.
One-tenth volume was removed for DNA determination.
J
I
0
r?
P
Y
-
I
I
I
I
2
3
4
5
6
7
8
9
10
I
rec y-IFN Concentration (units/ml x lo2)
Figure 1. Dose-dependent inhibition of progressive systemic sclerosis (PSS) fibroblast collagen synthesis by recombinant -yinterferon (rec y I F N ) . Confluent cultures of 2 PSS cell lines (GR
and SO) were incubated with increasing concentrations of rec y I F N
as described in Patients and Methods. Collagen production was
assessed after labeling with ''C-proline, by assaying aliquots of the
culture media for their L4C-hydroxyprolinecontent. Each value
shown1 is an average of 2 separate cultures.
853
Table 2. Effects of recombinant y-interferon (rec y-IFN) on
''C-hydroxyproline production by fibroblasts from patients with
progressive systemic sclerosis*
''C-hydroxyproline (dpm x
Media
+ 500 unitslml
rec y-IFN
Cell line
Media alone
KA
17.62
6.87
-61.0
so
33.48
29.07
25.62
5.82
4.96
3.69
-82.6
-82.9
-85.6
GR
21.01
18.67
16.58
4.08
2.94
3.16
-80.6
-84.3
-80.9
PR
16.66
2.44
-85.4
13.22
4.32
-67.3
26.72
10.99
-58.9
SB
% change
* Confluent monolayer cultures of dermal fibroblasts were incubated
with rec y-IFN for 48 hours as described in Patients and Methods, and
were labeled with I4C-prolinefor an additional 24 hours to determine
the production of newly synthesized collagen. All experiments were
performed in duplicate, and the values shown are the average of 2
determinations which differed from each other by <15%.
Total fibroblast RNA was then isolated using the
CsC12 discontinuous gradient method outlined by Maniatis
et a1 (12).For dot-blot analysis, amounts of total RNA equal
to 0.125, 0.5, and 2 pg were aliquoted into separate Eppendorf tubes. The samples were then incubated at 65°C for
3 minutes in a 3:l (vo1ume:volume) mixture of 100%
formamide, formaldehyde, and lox morpholinopropane
sulfonic acid (MOPS) (200 mM MOPS, 10 mM EDTA, 50
mM sodium acetate, pH 7.0) and placed into an ice slurry.
The contents of each tube were then brought to a salt
concentration equal to lox standard saline citrate (SSC),
and samples containing various amounts of RNA were
blotted onto nitrocellulose (preequilibrated with lox SSC)
using an S & S dot-blot apparatus as described by Kafotos et
a1 (13). Hybridization to the filter-bound nucleic acid was
performed as previously described (14).
Each of the RNA samples was analyzed in duplicate
at each of 3 concentrations. Autoradiographs of RNA dotblots were analyzed using a densitometer, and the areas
under the obtained peaks were quantified with the use of a
planimeter. The areas under the peaks were linear with
respect to the quantities of total RNA hybridized.
The complementary DNA (cDNA) probes used in
these analyses, Hf677 and E6, have been previously isolated
and characterized (14-16). These probes are specific for
al(1) and al(II1) human procollagen genes.
RESULTS
Collagen production by PSS cell lines. All cell
lines utilized for these studies displayed increased
ROSENBLOOM ET AL
854
Figure 2. Dot-blot hybridization of messenger RNA from control and recombinant yinterferon (rec
y1FN)-treated progressive systemic sclerosis fibroblast cultures to human complementary DNA (cDNA)
probes for ( ~ l ( 1 and
) (u1(III) procollagen chains. Total RNA obtained from cell line SO fibroblasts, untreated
(control) or treated with 500 units/ml of rec y I F N , was hybridized to the human cDNA clones for pro-al(1)
(clone Hf677) and pro-al(1II) (clone E6) collagen chains, as described in Patients and Methods. After
scanning densitometry analysis of the blots, the areas under each peak were determined using a planimeter.
.- 0.2-
/
/
/
u
.d
1
3
w
I
E6
TOTAL RNA HYBRIDIZED (pg)
Figure 3. Plot of the intensity of dot-blots obtained in messenger
RNA (rnRNA) hybridization experiments (see Figure 2) versus the
concenlration of mRNA hybridized. Values shown represent the
average of 2 separate determinations at each concentration of RNA
applied The calculated slopes of each line represent the amount of
mRNA specific for each collagen transcript per pg of total RNA.
Open circles = untreated cells; closed circles = treated cells.
collagen production compared with control cell lines,
as shown in Table 1. The average collagen production
by the PSS cell lines studied was 117% greater than the
average for the normal cell lines. Furthermore, some
PSS cell lines (SO, GR, and SB) produced 2-3-fold more
collagen than did their matched normal counterparts.
Effects and dose-response of rec pIFN on PSS
fibroblast collagen synthesis. The effects of various
concentrations of rec y I F N , ranging from 0--1,000
units/ml, on 14C-hydroxyprolineproduction by 2 PSS
cell lines were examined. The results, shown in Figure
1, demonstrate a concentration-dependent inhibition
of 14C-hydroxyproline production by the PSS fibroblasts. Near maximal inhibition was attained with 500
unitslml of rec y I F N ; therefore, this concentration
was used in all subsequent studies.
Table 2 shows the effects of 500 units/ml rec
y I F N on collagen production by confluent cultures of
the 5 PSS cell lines. The results demonstrate that the
rec y I F N caused a reproducible inhibition of 14Chydroxyproline production by the 5 cell lines, ranging
from 58.9% to 85.6%. At the concentrations used in
this study, rec y I F N did not have a significant effect
on PSS fibroblast viability or proliferation, since the
number of viable fibroblasts present in the cell layers
of parallel cultures incubated with control medium and
those incubated with medium containing 500 units/ml
IFN MODULATION OF PSS FIBROBLAST COLLAGEN PRODUCTION
Table 3. Relative concentrations of procollagen messenger
RNAs in untreated fibroblasts from progressive systemic sclerosis
(PSS) patients and in PSS fibroblasts treated with recombinant
y-interferon*
-
Untreated
Treated
% change
-
Collagen-specific RNA
(cm2/mg DNA)
pro-al(1)
pro-al(II1)
97.4
28.9
-70.3
156.8
57.4
-63.4
* Messenger RNA analyses were performed on total RNA from cell
line S O fibroblasts by dot-blot hybridization utilizing specific complementary DNA probes, as described in Patients and Methods. The
dot-blots were scanned in a densitometerafter autoradiography, and
the areas under each peak were calculated. The values shown were
calculated from the area under each peak/jg RNA, using the
equation:
Collagen-specific RNA (cm*/mg DNA) =
(Integrated area of hybridized RNA/pg RNA) x total RNA in culture flask
Total mg DNA in culture flask
rec y I F N were not statistically different (data not
showin).
Quantitation of type I and type 111 procollagen
mRN14levels by dot-blot hybridization. We have previously shown that rec y I F N decreased the steadystate level of al(1) procollagen mRNA in normal
human cultured fibroblasts (3). In order to determine
whether rec y I F N exhibited a similar effect on PSS
fibroblasts and whether there was coordinate regulation of types I and I11 procollagens, the levels of
mRNA for collagen chains al(1) and al(II1) were
deterimined by dot-blot hybridization, using cloned
cDNA probes specific for these 2 chains. Because of
the limited availability of rec y IF N , only the PSS cell
line that exhibited the highest level of collagen synthesis (SO) was selected for these detailed studies. The
results, presented in Figures 2 and 3 and in Table 3
show that treatment with 500 unitdm1 of rec y I F N
produced a 70% and 63% decrease, respectively, in
al(1) and al(II1) procollagen mRNA levels, relative to
untreated cells. These results suggest that there is
coordinate regulation of type I and type I11 procoflagen mRNA levels by rec y I F N and that the inhibition of PSS fibroblast collagen production can be
explained largely by the decreased levels of these
mRNAs.
DISCUSSION
The results presented here demonstrate that rec
y IF14 is capable of influencing the biosynthetic behavior of PSS fibroblasts in a manner similar to that
855
previously described for normal fibroblasts (2-5). The
inhibition of PSS fibroblast collagen production by rec
y I F N was concentration-dependent (Figure 1). When
the effects of 500 units/ml of rec y I F N on collagen
biosynthesis by PSS fibroblasts were examined, it was
found that the rec y I F N consistently caused a striking
inhibition of collagen production by the 5 cell lines
studied (Table 2). The inhibition of collagen production ranged from 58.9% to >85%. These results are
more remarkable when one considers that all the cell
lines studied were obtained from patients with rapidly
progressive disease. Furthermore, all the fibroblasts
tested exhibited increased collagen production in culture when compared with that in normal fibroblasts
(Table 1).
The results of the hybridization experiments
with the specific probes for al(1) and al(II1) procollagen chains clearly demonstrated that rec yI F N
caused a coordinate decrease of >60% in the mRNA
levels for these chains (Figures 2 and 3 and Table 3).
These results strongly suggest that the decrease in PSS
fibroblast collagen production is a result of this corresponding decrease in type I and type I11 procollagen
mRNA levels. Since, in the majority of cases, such
alterations in mRNA are due to changes in transcription rates, it is likely that y I F N decreases the rate at
which the al(I> and al(II1) procollagen genes are
transcribed. Further experiments are needed, however, to determine whether this is the sole explanation
for these findings or whether other factors, such as
changes in mRNA processing or stability, occur.
The frequent histopathologic observation of
mononuclear cell infiltration in close proximity to
fibroblasts in areas of early fibrosis (17,18), coupled
with the demonstration that rec y I F N is capable of
potent inhibition of collagen production by PSS fibroblasts, suggest that mononuclear cells from PSS patients may have a defect in the production of yI F N,
and therefore may not be able to modulate the expected fibroblastic response occurring in areas of
immunologically mediated inflammation. This possibility, which may have relevance regarding the pathogenesis of fibrosis in PSS, is currently being explored.
The potent inhibition of PSS fibroblast collagen
production by rec y I F N suggests the feasibility of a
novel therapeutic approach to PSS: the use of yI F N
to modulate the excessive PSS fibroblast collagen
production. These findings are encouraging since, at
the present time, there is no treatment which can
effectively prevent the frequently progressive fibrosis
that is characteristic of this disease.
ROSENBLOOM ET AL
856
ACKNOWLEDGMENTS
We thank Dr. Jeanne Myers for providing the human
cDNA clones. We gratefully acknowledge the technical
assistance of Ruth Herold, Mary Dixon, and Ronald
Yankowski. The expert assistance of Esther Lobb in the
preparation of the manuscript is appreciated.
REFERENCES
1. LeRoy EC: Scleroderma (systemic sclerosis), Textbook
of Rheumatology. Second edition. Edited by WN
Kelley, ED Harris Jr, S Ruddy, CB Sledge. Philadelphia, WB Saunders, 1985, pp 1183-1205
2. Jimenez SA, Freundlich B, Rosenbloom J: Selective
inhibition of human diploid fibroblast collagen synthesis
by interferons. J Clin Invest 74:1112-1116, 1984
3. Rosenbloom J, Feldman G , Freundlich B, Jimenez SA:
Transcriptional control of human diploid fibroblast
collagen synthesis by y-interferon. Biochim Biophys
R'es Commun 123:365-372, 1984
4. Slephenson ML, Krane SM, Amento EP, McCroskery
PA, Byrne M: Immune interferon inhibits collagen synthesis by rheumatoid synovial cells associated with
decreased levels of the procollagen mRNAs. FEBS Lett
1880:43-50, 1985
5. Duncan MR, Berman B: y interferon is the lymphokine
anid a interferon the monokine responsible for inhibition
of fibroblast collagen production and late but not early
fibroblast proliferation. J Exp Med 162516527, 1985
6. Rubinstein S, Familletti PC, Pestka S: Convenient assay
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7. Subcommittee for Scleroderma Criteria of the American
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Criteria Committee: Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis
Rheum 23:581-590, 1980
8. Bashey RI, Jimenez SA: Increased sensitivity of
scleroderma fibroblasts to stimulation of protein and
collagen synthesis by serum. Biochim Biophys Res
Commun 76: 1214-1222, 1977
9. Brouty-Boye D: Inhibitory effects of interferons on cell
multiplication. Lymphokine Res 1:99-110, 1980
10. Juva K, Prockop DJ: Modified procedure for the assay
of [3H]- or [I4C]-labeled hydroxyproline. Anal Biochem
15:77-80, 1966
11. Jimenez SA, McArthur W, Rosenbloom J: Inhibition of
collagen synthesis by mononuclear cell supernatants. J
Exp Med 150:1421-1431, 1979
12. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning. Cold Spring Harbor, NY, Cold Spring Harbor
Laboratory, 1982, pp 196-199
13. Kafotos EC, Jones CW, Efstratiadis A: Determination
of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic
Acids Res 7:1541-1552, 1979
14. Myers JC, Chu ML, Faro SH, Clark WJ, Prockop DJ,
Ramirez F: Cloning a cDNA for the pr0-a2 chain of
human type I collagen. Proc Natl Acad Sci USA
78:35 16-3520, 1981
15. Chu ML, Myers JC, Bernard MP, Ding JF, Ramirez F:
Cloning and characterization of five overlapping cDNAs
specific for the human pro-a, (I) collagen chain. Nucleic
Acids Res 105925-5934, 1982
16. Loidl H-R, Brinker JM, May M, Pihlajaniemi T, Morrow
S, Rosenbloom J , Myers JC: Molecular cloning and
carboxyl-propeptide analysis of human type 111 procollagen. Nucleic Acids Res 12:9383-9394, 1984
17. Arey LB: Wound healing. Physiol Rev 16:327-338, 1936
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13:104-113, 1983
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