From the Division of Immunology and Allergy, Clinical Immunology Unit (Hans Wilsdorf Laboratory), Department of Internal Medicine, University Hospital, Geneva, Switzerland
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ABSTRACT |
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In many inflammatory diseases where tissue
remodeling occurs, T cells are in close contact with mesenchymal cells.
We investigated the effect of direct cell-cell contact between
peripheral blood T lymphocytes or HUT-78 lymphoma cells and dermal
fibroblasts or synoviocytes on the deposition of the major
extracellular matrix components: types I and III collagen. Incubation
of dermal fibroblasts and synoviocytes with plasma membrane
preparations from resting T cells slightly increased the production of
collagen I but did not significantly affect that of collagen III.
Conversely, direct contact with either plasma membranes or fixed
phytohemagglutinin/phorbol myristate acetate-activated T cells markedly
inhibited the synthesis of types I and III collagen by 60-70% in
untreated dermal fibroblasts and synoviocytes and by 85% in
transforming growth factor -stimulated fibroblasts. This decrease of
collagen synthesis was abrogated when fixed T cells were separated
physically from fibroblasts, demonstrating that direct contact between
the two cell types was necessary. This inhibition was associated with a
marked decrease in steady-state levels of pro-
1(I) and pro-
1(III)
collagen mRNAs. T cell contact decreased the transcription rate but
did not significantly alter the stability of the
1(I) and
1(III)
transcripts. Finally, using neutralizing antibodies or cytokine
inhibitors we provide evidence that this inhibition of extracellular
matrix production mediated by T cell contact was partially due to
additive effects of T cell membrane-associated interferon
, tumor
necrosis factor
, and interleukin-1
.
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INTRODUCTION |
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Regulation of connective tissue metabolism is an important event in a number of biological and pathophysiological processes such as embryonic organomorphogenesis, wound healing, inflammation, tissue destruction, fibrosis, tumor invasion, and metastasis. Fibroblasts are mesenchymal cells which play a crucial role in the remodeling of extracellular matrix (ECM)1 by synthesizing and organizing connective tissue components, predominantly constituted of types I and III fibrillar collagens. Type I collagen is abundant in the skin, tendons, and bones and it is also the main collagen type produced by dermal fibroblasts in culture (1, 2).
Fibroblasts respond to various microenvironmental signals including soluble cytokines and growth factors as well as cell-matrix or cell-cell interactions which intervene notably in the control of the balance between synthesis and degradation of ECM (3). Alterations in this balance can lead to pathological events such as the invasion of tissue by malignant cells (4), abnormal ECM deposition in fibrotic diseases (5) or, conversely, to tissular destruction in chronic inflammation (6). Furthermore, in some pathological conditions there is evidence for a lack of ECM neosynthesis (i.e. proteoglycans) and consequently a lack of repair (7, 8).
It is highly suggested that T cells may play an important role in the pathogenesis of some chronic inflammatory diseases (i.e. rheumatoid arthritis, scleroderma) not only through the release of soluble factors but also through direct contact with fibroblasts or fibroblast-like cells (i.e. synoviocytes) (9-13). This contact can induce the production of cytokines, matrix metalloproteinases (MMP), prostaglandins (PGE2) and control the expression of adhesion molecules, substantiating the assumption that the interaction between fibroblasts and inflammatory cells might influence both fibroblast activation and inflammatory response (14-18).
A wide range of cytokines exert profound effects on fibroblast
migration, proliferation, and ECM production. Transforming growth
factor- (TGF
), IL-4, IL-6, IL-13, platelet-derived growth factor,
epidermal growth factor, and basis fibroblast growth factor are
fibrogenic cytokines and growth factors while IFN-
, IFN-
, IFN-
, IL-10, relaxin, and leukoregulin suppress collagen synthesis (5, 19-26). The effects of TNF
and IL-1 on the production of collagen by fibroblasts appear much more controversial and depend on
the cell type (19, 27-30).
Collagen is known to control its own synthesis by fibroblasts through a
negative feedback loop via interaction with the 1
1 integrin (31,
32). In contrast to these well documented fibroblast-ECM interactions,
little information is available concerning the role of cell-cell
contact in the regulation of ECM deposition. Previous in
vitro studies suggest that the interaction between inflammatory cells and fibroblasts can modulate several fibroblast functions including collagen production (33-35). However, these studies do not
distinguish the effects of monocytes/macrophages from the action of T
lymphocytes. Nor do they take into account the respective role of
soluble factors released by mononuclear cells and those requiring
direct cell-cell contact in the mediation of this phenomenon.
The aim of the present study was to shed light on the regulation of ECM deposition, mainly types I and III collagen, operated by direct cell-cell interaction between inflammatory cells, namely T lymphocytes or for convenience and standardization the HUT-78 T lymphoma cell line, and mesenchymal cells such as dermal fibroblasts and synoviocytes.
We provide evidence that the physical interaction between
PHA/PMA-activated T cells and dermal fibroblasts or synoviocytes reduces markedly the production of both types I and III collagen by
these cells. We also demonstrate that this inhibition is regulated at
the transcriptional level and that it is mainly due to an additive effect of T cell-associated IFN-, TNF
, and IL-1
.
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EXPERIMENTAL PROCEDURES |
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Reagents--
Dulbecco's modified Eagle's medium, RPMI 1640 medium, phosphate-buffered saline, penicillin, streptomycin,
L-glutamine were supplied by Life Technologies (Paisley,
United Kingdom) and fetal calf serum from Seromed (Biochrom KG, Berlin,
Germany). Cycloheximide (CHX), 5,6-dichlorobenzimidazole riboside
(DRB), -aminopropionitrile,
-ketoglutaric acid,
L-ascorbic acid, iodoacetamide, indomethacin, and PMA
(phorbol myristate acetate) were purchased from Sigma. [
-32P]dCTP (3000 Ci/mmol) and
[
-32P]UTP (3000 Ci/mmol) were from Hartmann Analytic
Gmbh (Braunschweig, Germany). Paraformaldehyde was from Merck
(Darmstadt, Germany). Phaseolus vulgaris
leucophytohemagglutinin (PHA) was from E-Y Laboratories Inc. (San
Mateo, CA). Human recombinant TGF
(hTGF
1) was from R & D Systems
(Minneapolis, MN). IFN-
, TNF
, and IL-1
were from Biogen
(Geneva, Switzerland). Human recombinant soluble TNF receptor p55
(rsTNF-p55-h
3) was the kind gift of H. Loetscher (Hoffmann-La Roche,
Basel, Switzerland). Human recombinant IL-1Ra was obtained from
Synergen (Boulder, CO). The anti-IFN
mAb was generously provided by
Dr. G. Garotta (Hoffmann-La Roche, Basel, Switzerland).
Dermal Fibroblasts and Synoviocytes-- Human dermal fibroblasts and synoviocytes were isolated by protease treatment of foreskin and surgical synovectomy specimens from rheumatoid arthritis patients, respectively, as described previously (36). Synoviocytes were used between the fourth and tenth passages. Dermal fibroblasts and synovial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 50 µg/ml streptomycin, 50 IU/ml penicillin, and 2 mM L-glutamine at 37 °C in 5% CO2. For contact experiments fibroblasts were plated in flat-bottom 96-well tissue culture trays (Costar) at 2 × 104 cells/well in complete Dulbecco's modified Eagle's medium.
T Cells and T Cell Line-- Peripheral blood T lymphocytes (PBTL) were purified from buffy coats of healthy donors as described previously (37). They contained 94-98% CD2+, 83-94% CD3+, and <2% CD14+, i.e. less than 2% of monocytes as assessed by flow cytometry. The HUT-78 human cutaneous T lymphoma cell line (38) was obtained from ATCC (Rockville, MD). T cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml streptomycin, 50 IU/ml penicillin, and 2 mM L-glutamine in 5% CO2-air humidified atmosphere at 37 °C.
Stimulation, Fixation, and Membrane Preparation of T Cells-- HUT-78 cells (1 × 106 cells/ml) and PBTL (4 × 106 cells/ml) were cultured for the indicated times in complete RPMI medium at 37 °C in the absence or presence of 1 µg/ml PHA and 5 ng/ml PMA. To avoid complications due to the presence of two viable cell types in the culture medium during cell-cell contact experiments, resting or stimulated T cells were washed thoroughly in phosphate-buffered saline and either fixed with 1% paraformaldehyde or resuspended for plasma membrane preparation (37).
For plasma membrane preparation, T cells were broken by sonication (five 5-s bursts of 90 W each) in phosphate-buffered saline containing 0.68 M sucrose, 200 µM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 0.1 µg/ml pepstatin, and 5 mM EDTA. The lysate was centrifuged for 15 min at 4,000 × g to discard nuclei and unbroken cells. The supernatant was centrifuged for 45 min at 100,000 × g and the pellet containing the membrane fraction was resuspended by sonication at the theoretical concentration of 50 × 106 cell equivalent/ml in phosphate-buffered saline, 20 µM EDTA, 5 µM iodoacetamide.Cell-to-cell Contact Experiments--
Since in many cell systems
collagen expression varies with the stage of growth (39, 40)
experiments were performed on confluent fibroblast cultures prepared at
least 48 h before contact. After removing the medium, fixed T
cells or T cell membranes (2-5 × 105 cell
equivalent/well) were incubated for 48 h with dermal fibroblasts or synoviocytes in a final volume of 200 µl/well of Dulbecco's modified Eagle's medium supplemented with 1% FCS, 50 µg/ml
-aminopropionitrile, 3.5 µg/ml
-ketoglutaric acid, and 25 µg/ml L-ascorbic acid. The culture supernatants were
frozen at
20 °C for further determination of types I and III
collagen contents.
Determination of Types I and III Collagen Production-- The production of types I and III collagen was estimated in 48 h culture media by measuring the concentration of the amino-terminal propeptides of procollagen I and III (PINP and PIIINP) by competition-based radioimmunoassays (Orion Diagnostica, Espoo, Finland). The threshold of the radioimmunoassays for PINP and PIIINP were 2 and 0.2 ng/ml, respectively.
RNA Extraction and Northern Blot Analysis--
Total RNA was
isolated from confluent fibroblast monolayer cultures (in 60-mm Petri
dishes) by lysing the cells with TRIzolTM reagent (Life
Technologies) according to the manufacturer's procedures. RNAs (5-10
µg) were separated by electrophoresis in 1% formaldehyde agarose
gel, transferred onto nylon Hybond N membrane (Amersham), and
hybridized to 32P-labeled cDNA probes specific for
pro-1(I) collagen (Hf677) (41), pro-
1(III) collagen
(Hf934) (42), pro-
2(I) collagen (Hf1131) (43), MMP-1 (44),
and GAPDH (45). Autoradiographs were quantified by densitometric
scanning using a laser densitometer equipped with ImageQuant software
(Molecular Dynamics) and values were normalized to GAPDH signals.
Nuclei Isolation and Run-on Transcription Assay--
Preparation
of nuclei from dermal fibroblast (2 × 107 cells),
transcription assay, and hybridization were performed as described previously (46). To ensure that comparable amounts of nuclei would be
present in each condition, the DNA content in lysed aliquots was
determined. Biosynthetically radiolabeled mRNAs (5 × 106 cpm) were hybridized onto slot-blotted cDNA (4 µg/slot of linearized Hf677, pBSGAPDH, or pBR322 as a control) for
48 h at 65 °C in 6 × SSC, 0.5% SDS, 0.1%
polyvinylpyrolidone, 0.1% Ficoll, 50 mM sodium phosphate,
pH 6.5, and 100 µg/ml denatured salmon sperm DNA, washed, and treated
with RNase A. Filters were dried and exposed to Amershan Hyperfilms MP
at 80 °C.
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RESULTS |
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Freshly Isolated Peripheral Blood T Lymphocytes and HUT-78 T Lymphoma Cells Modulate Types I and III Collagen Production during Direct Cell-Cell Contact with Human Dermal Fibroblasts and Synoviocytes-- The role of direct cell-cell interactions between resting or activated T lymphocytes and dermal fibroblasts or synoviocytes on collagen deposition was assessed by determining the concentration of NH2-terminal propeptides of procollagens I and III (PINP and PIIINP) in culture supernatants.
Confluent dermal fibroblasts and synoviocytes constitutively produced type I collagen at 63 ± 25 (n = 24) and 90 ± 29 ng/ml (n = 8), respectively, and to a lower extent type III collagen at 13.6 ± 2.8 and 18.7 ± 5.5 ng/ml, respectively. Co-cultures of dermal fibroblasts or synoviocytes and paraformaldehyde-fixed unstimulated PBTL or HUT-78 cells slightly increased the production of type I collagen but did not significantly affect type III collagen production (Fig. 1). Conversely, incubation with fixed PHA/PMA-activated PBTL or HUT-78 cells markedly decreased types I and III collagen production both in dermal fibroblasts and synoviocytes. Interestingly, in all experiments the inhibition of PINP and PIIINP production mediated by cell-cell contact was more efficient than that induced by a potent soluble anti-fibrogenic effector such as interferon-
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Direct Contact with Activated T Cells Decreases Steady-state Levels
of Procollagen I and III mRNAs in Both Human Dermal Fibroblasts and
Synoviocytes--
To further investigate the molecular mechanisms
underlying the inhibition of types I and III collagen, we focused on
the regulation of procollagen mRNAs by cell-cell contact. For this
purpose dermal or synovial fibroblasts were incubated for 14 h
with TGF or membranes of unstimulated or activated PBTL and HUT-78
cells, and expression of
1(I),
2(I), and
1(III) collagen chain
genes was estimated by Northern blot hybridizations with specific
probes.
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Cell Contact Inhibited the Transcription of Pro-1(I)
Collagen--
In order to determine the mechanism underlying the
inhibition of procollagen I transcripts level we measured the effect of cell contact on the transcription of pro-
1(I) collagen gene. Nuclei
were isolated from dermal fibroblasts cultured for 4 h in the
absence or presence of membranes from unstimulated or PHA/PMA-activated HUT-78 cells, and run-on experiments were performed. According to the
results presented in Fig. 6, the relative
de novo mRNA synthesis of pro-
1(I) collagen was
increased 1.5-fold in dermal fibroblasts treated with unstimulated
HUT-78 cells. Conversely, this transcription rate was decreased 4-fold
in dermal fibroblasts incubated with PHA/PMA-activated HUT-78 cells.
These data are in total agreement with results obtained from Northern
blots and thus imply that contact-induced down-regulation of collagen I production was controlled by a transcriptional mechanism sufficient to
result in a 4-fold reduction in pro-
1(I) collagen mRNA level in
dermal fibroblasts.
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Role of Membrane-associated Cytokines in the Down-regulation of
Type I Collagen Production--
In order to identify the molecules
present on activated T cells and likely to control the down-regulation
of collagen I production, we first used blocking mAbs raised against
CD2, CD11a, CD11b, CD11c, CD18, CD40, CD54, CD58, and CD106. Since
these antibodies failed to reverse the inhibition of collagen I
synthesis (not shown), we next tested the effect of specific inhibitors
or neutralizing mAbs raised against cytokines (IFN-, TNF
, and
IL-1), known to decrease in vitro the production of collagen
I by dermal fibroblasts. The blocking agents used in these experiments
were IL-1 receptor antagonist (IL-1Ra), human recombinant soluble TNF
receptor p55 (rsTNF-p55-h
3), and a neutralizing anti-IFN
monoclonal antibody.
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DISCUSSION |
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In the present study we investigated the effect of direct
cell-cell interactions on the production of types I and III collagen by
human dermal fibroblasts and synoviocytes. We demonstrate that co-culturing fixed PHA/PMA-activated PBTL or HUT-78 lymphoma cells with
dermal fibroblasts or synoviocytes results in a marked decrease in the
basal production of types I and III collagen. In contrast, contact with
unstimulated fixed T cells slightly increased the production of
collagen I while that of collagen III was not significantly affected.
These results suggest that cell-cell contact modulates connective
tissue metabolism. However, it is well established that soluble factors
may be released by fixed T cells. Thus, authors have shown that
significant levels of TNF (4-234 pg/ml) can be detected in culture
supernatants from fixed T cells (48). To rule out the possible
involvement of signals generated by both cell-cell contact and soluble
factors in the regulation of collagen synthesis, experiments were set
up first using plasma membrane preparations from unstimulated or
activated T cells, and then in double-chamber culture systems. Results
showed that contact-mediated signals are responsible for the inhibitory
action of activated T cells on the production of collagen I and III,
while diffusing mediators released from unstimulated T cells slightly
up-regulate collagen I synthesis.
Using specific neutralizing antibody we identified TGF as the
diffusing factor responsible for the increase in collagen I production
mediated by unstimulated T cell membranes or fixed cells (not shown).
This could explain why collagen III production is not up-regulated by
incubation with resting T cells since in this system production of type
III collagen appeared less sensitive to TGF
than did that of
collagen I.
In vitro studies revealed that human T cells have the
ability to adhere to synoviocytes as well as to dermal fibroblasts via CD2/CD58 (LFA-3) and LFA-1 (CD11a/CD18)/CD54 (ICAM-1) interactions (12,
13, 49). Furthermore, the cellular adhesion between T cells and
synoviocytes induces the production of IL-1 by synovial cells in
part through LFA-1-ICAM-1 interaction (17). Consequently, the putative
implication of these adhesion molecules in the inhibition of collagen I
production mediated by contact with activated T cells was investigated.
In the presence of blocking mAbs raised against CD2, CD11a, CD11b,
CD11c, CD18, CD40, CD54, CD58, and CD106, the decrease of collagen I
production was not reversed (data not shown), thus ruling out the
involvement of these surface molecules in the mediation of cell-contact
mediated inhibition of collagen synthesis.
Next, we investigated the implication of cell-associated cytokines on
the inhibition of collagen deposition. Several cytokines are known to
decrease in vitro the production of collagen I by fibroblasts i.e. IFN-, TNF
, and in some cases IL-1
and IL-1
(29, 30, 50). Moreover, membrane-associated TNF
expressed by activated T cells has been shown to provide stimulatory
signals for the activation of human B cells, monocytes, and endothelial cells (51-53). Membrane-associated IL-1 has also been described (54),
and detectable levels of IL-1
and TNF
measured in plasma membrane
preparations of PHA/PMA-activated PBTL have been shown to stimulate
MMP-1 and PGE2 production by dermal fibroblasts and synoviocytes (18). Furthermore, it has recently been reported that
IFN
is expressed on the surface of Th1 cells (55). Therefore, we
used antibodies or cytokine inhibitors for these cytokines and
demonstrate that the concomitant neutralization of IFN-
, TNF
, and
IL-1 results in a marked reversion of the inhibition of collagen I
production amounting to 81 and 66.7% in dermal fibroblasts cultured
with activated PBTL and HUT-78 cells, respectively. Thus, the
inhibition of collagen production seems to be mainly due to activated T
cell-associated cytokines including IL-1
, IFN-
, and the
transmembrane form of TNF
. However, since cytokine blocking agents
did not prompt the complete reversion of collagen down-regulation, particularly in the case of activated HUT-78 cells, we propose that
other still unidentified cell-surface molecules might be implicated in
this inhibitory effect. Besides, since IL-1
was only detected in the
membrane preparations of activated PBTL and not in those of HUT-78
cells, we cannot rule out the possible implication of a cytokine
autocrine loop produced by fibroblasts following contact with activated
PBTL or HUT-78 cell membranes. Indeed, co-cultures of T cells and
dermal fibroblasts or synoviocytes have been reported to stimulate the
production of IL-1
by fibroblasts (16, 17), and Bombara et
al. (14) have reported that co-culturing T cells and synovial
fibroblasts results in the accumulation of cytokines in the culture
supernatant, notably of IFN-
and TNF
(14). However, according to
RNase protection assay analysis, in our system mRNAs encoding for
IFN-
and TNF
were not expressed in dermal fibroblasts or
synoviocytes incubated with membranes of activated T cells (not shown).
These data confirm that inhibition of collagen production mediated by
IFN-
and TNF
is due to membrane associated forms of these
cytokines.
The involvement of membrane-associated cytokines in the down-regulation
of collagen deposition is consistent with the time course of induction
by PHA/PMA of the ability of PBTL to inhibit the production of
collagen. Indeed, TNF which is the best characterized membrane-associated cytokine, was shown to be expressed on the surface
of CD4+ T cell clones within 2 h after activation
(51).
Previous reports have shown that soluble IFN- and TNF
inhibit
collagen synthesis both by transcriptional and post-transcriptional mechanisms (28, 56). We demonstrate here that contact-mediated decrease
of types I and III collagen takes place exclusively at the
transcriptional level. Indeed, we provide evidence that no post-transcriptional modifications likely to destabilize pro-
1(I) and pro-
1(III) mRNAs take place. Furthermore, this inhibition was specific for collagen genes since the expression of mRNA coding for MMP-1 was up-regulated during T cell-fibroblast interaction. However, the inhibition of types I and III collagen probably involves distinct molecular mechanisms. Indeed, the decrease of pro-
1(I) mRNA required de novo protein synthesis whereas that of
pro-
1(III) did not.
Finally, having observed that direct contact with activated T cells
induced the production of PGE2 in dermal fibroblasts (18), and PGE2 having proved to inhibit fibroblast collagen
synthesis (57), we studied the effect of activated T cell membranes on collagen I production in the presence of indomethacin. The latter, however, did not reduce the decrease of collagen I production mediated
by contact with activated T cells (not shown). These data match
previous reports on the inhibitory action of soluble IL-1, TNF, and
IFN-
on collagen I and III accumulation in lung fibroblasts (58).
Consequently, the additive inhibitory effect of cell-associated TNF
,
IFN-
, and IL-1 on collagen I and III production by dermal
fibroblasts and synoviocytes takes place at the transcriptional level
by a PGE2-independent mechanism.
Our observations demonstrate for the first time to our knowledge that
collagen production might be regulated in vivo by close contact between mesenchymal cells and T lymphocytes. This work also
indicates that membrane-associated IFN- might be biologically active
as already described for surface TNF
. The nature of the binding of
IFN-
to activated T cell membranes is still unclear. However, as
proposed by Assenmacher et al. (55) it would not be IFN-
bound to IFN
R since surface-bound IFN-
was detected with several
mAbs which block the binding of mouse IFN-
to its receptor. Further
experiments are at present being conducted in our laboratory using
CD4+/CD8+ as well as Th1 and Th2 T cell clones
activated by an immobilized anti-CD3 antibody to specify the role of
membrane-associated IFN-
in the contact-mediated inhibition of
collagen production. Current works are also under progress to study
this contact-mediated inhibition of collagen deposition in diffuse
systemic scleroderma, a fibrotic disease characterized by abnormal
deposition of collagen. In these conditions, the decrease of type I
collagen seem to be significantly less pronounced in fibroblasts from
systemic scleroderma patients than normal individuals, suggesting that
resistance of systemic scleroderma fibroblasts to inhibition might play
a pathogenic role in systemic scleroderma (59).
In conclusion, remodeling of ECM occurs in many biological processes and involves the controlled degradation and neosynthesis of collagenous and non-collagenous components. Types I and III collagen are the main constituents of connective tissue and their homeostasis is finely regulated by various signals including soluble factors, cell-matrix, and cell-cell interactions (5). We have reported that direct contact between activated T cells and dermal fibroblasts or synoviocytes induced an imbalance between the production of interstitial collagenase (MMP-1) and that of tissue inhibitor of metalloproteinases-1 (TIMP-1), favoring matrix catabolism (18). We demonstrate here that this contact appears to be also capable of favoring ECM degradation by decreasing the level of synthesis of the main collagenous components of ECM, which might reinforce the tissular destruction effect and the lack of repair in vivo.
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ACKNOWLEDGEMENTS |
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We express our sincere thanks to Rachel Chicheportiche and Marie-Thérèse Kaufmann for skilful technical assistance.
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FOOTNOTES |
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* This work was supported by Swiss National Science Foundation Grant 31-50930-97.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Hôpital Cantonal
Universitaire de Genève, Unité d'Immunologie Clinique,
1211 Genève 14, Switzerland. Tel.: 41-22-372-91-94; Fax:
41-22-372-93-69; E-mail: Rezzonico-roger{at}diogenes.hcuge.ch.
1 The abbreviations used are: ECM, extracellular matrix; PBTL, peripheral blood T lymphocytes; MMP, matrix metalloproteinases; PINP, procollagen I NH2-terminal propeptide; PIIINP, procollagen III NH2-terminal propeptide; DRB, 5,6-dichlorobenzimidazole riboside; CHX, cycloheximide; PGE2, prostaglandin E2; IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TGF, transforming growth factor; mAb, monoclonal antibody.
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REFERENCES |
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