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Stimulation of the secretion of latent cysteine proteinase activity by tumor necrosis factor ╨Ю┬▒ and interleukin-1.

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STIMULATION OF THE SECRETION OF
LATENT CYSTEINE PROTEINASE ACTIVITY BY
TUMOR NECROSIS FACTOR a! AND
INTERLEUKIN- 1
GUILLEMETTE HUET, RENE-MARC FLIPO, CECILE COLIN, ANNE JANIN, BRIGITTE HEMON,
MAUD COLLYN-D'HOOGHE, ROBERT LAFYATIS, BERNARD DUQUESNOY, and PIERRE DEGAND
Objective. Cultured synovial fibroblast-like cells
from 3 patients with rheumatoid arthritis (RA) and 3
patients with osteoarthritis (OA) were evaluated for
their potential to secrete cysteine proteinases spontaneously and after stimulation by tumor necrosis factor a
(TNFa) or interleukin-1 (IL-1).
Methods. Culture media and cell lysates were
analyzed before and after high performance liquid
chromatography (HPLC) using the enzymatic substrate,
Z-Phe-Arg-AMC, and by immunoblotting with anticathepsin B antiserum. Immunolocalization of cathepsin
B was studied on cell monolayers.
Results. Latent cysteine proteinase activity was
found to be secreted spontaneously by cultured synovial
fibroblast-like cells. This activity was increased after
treatment with either TNFa or IL-1. Stimulated protease activity was eluted by HPLC at a peak coincident
From the Departments of Biochemistry, Pathology, and
Rheumatology, CHRU, University of Medicine, Research Unit,
INSERM U16, Lille, France.
Supported by grants from DREDAJniversity of Lille 11,
ARP (Association de Recherche sur la Polyarthrite), and CHRU
Lille.
Guillemette Huet, MCU-PH: Department of Medicine,
CHRU Lille; R e d - M a r c Flipo, MD: Department of Rheumatology,
CHRU, University of Medicine, Lille; Ctcile Colin: Student, Research Unit, INSERM U16; Anne Janin, MD: Professor, Department of Pathology, CHRU, University of Medicine, Lille; Brigitte
Hemon: Technical Assistant, INSERM U16; Maud Collynd'Hooghe: ChargC de Recherche, INSERM U124, Lille; Robert
Lafyatis, MD: ChargC d e Recherche, INSERM U 167, Lille; Bernard
Duquesnoy, MD: Professor, Department of Rheumatology, CHRU,
University of Medicine, Lille; Pierre Degand, MD: Professor,
Department of Medicine, CHRU Lille.
Address reprint requests to Guillemette Huet, MCU-PH,
INSERM, Unit6 de Recherches no. 16, Biochimie des Proteines,
Place de Verdun, 59045 Lille Cedex, France.
Submitted for publication October 7, 1992; accepted in
revised form December 22, 1992.
Arthritis and Rheumatism, Vol. 36, No. 6 (June 1993)
with that of purified cathepsin B. By immunoblot, cell
supernatants contained a 43-kd form of cathepsin B,
while cell lysates contained a 30-kd form, consistent,
respectively, with cathepsin B before and after cleavage
of its propeptide. An intracellular increase in cathepsin
B after treatment with TNFa was also seen with immunohistochemical studies.
Conclusion. TNFa (in the 6 cases studied) and
IL-1 (in 4 cases) stimulated the secretion of a latent
cysteine proteinase activity from synovial fibroblast-like
cells, which appears to represent primarily cathepsin B.
Rheumatoid arthritis (RA) is a systemic disease
characterized by chronic joint inflammation that leads
to destruction of articular cartilage and periarticular
bone. Proteinases released in the joint are at least
partly responsible for the degradation of articular
connective tissues (1). Inflammatory synovial fluids
contain elevated levels of proteinases, such as elastase
(2-5), cathepsin G (2), collagenase (3,6), stromelysin
(6), and lysosomal cysteine proteinases (7-9). Other
than elastase and cathepsin G , which are released
primarily from neutrophils, many proteinases, including collagenase (lo), stromelysin (1 l), and lysosomal
proteinases (I), appear to be secreted by cells in the
rheumatoid synovium. Although elastase, collagenase,
and stromelysin have been the most thoroughly investigated proteinases that potentially contribute to cartilage degradation, several observations also suggest
potential roles for the lysosomal cysteine proteinases,
cathepsins B and L, in cartilage degradation.
Cathepsins B and L are able to degrade cartilage collagens types 11, IX, and XI (12). Cathepsin B
has been detected in rheumatoid synovial explants and
culture media (1 3). Van Noorden et a1 (1 4) have shown
TNFa AND IL-1 STIMULATION OF CYSTEINE PROTEINASE
that in arthritic knee joints of rats, there is a large
increase in cathepsin B activity in synoviocytes, chondrocytes, and fibroblasts, as well as extracellular
cathepsin B activity within the cartilage matrix. Treatment of some rats with Z-Phe-Ala-CH,F, the selective
inhibitor of cathepsin B, resulted in decreased cathepsin B activity and diminished cartilage damage. Cathepsin L has also been described in degradative processes of
cartilage and bone. In experimentally induced arthritis in
rabbits, Etherington e t al (15) have demonstrated
increased cathepsin L in the synovial lining.
In RA several cytokines appear t o have important roles in the regulation of the synovial cell phenotype. Interleukin-l (IL-l) and tumor necrosis factor a
(TNFa) augment the production of prostaglandin E,
(PGE,) and metalloproteinases, including collagenase
(16,17) and stromelysin (18). Platelet-derived growth
factor (PDGF) has been shown t o induce proliferation
and anchorage-independent growth of these cells (19).
Although IL-1p stimulates collagenase secretion by
rabbit articular chondrocytes, the cytokine does not
stimulate cathepsin B secretion t o a significant degree;
however, it does increase the intracellular pool of
cathepsin B by 2 4 - f o l d (20). IL-1 and TNFa can be
found in synovial fluids from RA and osteoarthritis
(OA) patients and are considered to be important
mediators in the pathogenesis of these diseases (21-23).
We studied the potential influence of TNFa and
1L-1 upon the secretion of cysteine proteinases by cultured synovial fibroblast-like cells derived from patients
with RA or OA. Our findings are presented herein.
PATIENTS AND METHODS
Patients. Synovial membrane samples were obtained
from patients undergoing surgery for total joint replacement.
Tissues were taken from 3 patients with RA and 3 with OA.
The RA patients met the American College of Rheumatology
(formerly the American Rheumatism Association) criteria
for moderate or severe disease (24). At the time of hospitalization, RA patients were taking a variety of antiinflammatory drugs.
Cell culture and cytokine treatment. Synovial fibroblast-like cells were obtained from synovial explants and
cultured directly in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 100 units/ml penicillin, 100
pg/ml streptomycin, 2.5 pglml amphotericin B, and 10%
heat-inactivated (56°C for 30 minutes) fetal calf serum (Boehringer Mannheim, Indianapolis, IN) at 37°C in humidified air
with 5% CO,. Synovial membranes removed aseptically
were rinsed in sterile saline solution. Connective tissue and
fat were discarded, and the synovial tissue was minced into
pieces smaller than 1 mm3 (with 2 scalpels). Several tissue
pieces were placed in each well of 6-well cluster plates
773
(Corning Glassware, Corning, NY) and incubated without
medium. After 1 hour, complete medium was carefully
added. Synovial fibroblast-like cells grew from the explants
within a week. Cells from passages 3 4 were used in all
experiments shown.
Cells were grown to near-confluency in 120-mm
plates or 6-well cluster plates. Treatment with IL-I (human,
natural, ultrapure grade containing 75% IL-lP and 25%
IL-la, specific activity lo8 unitshg of protein) or with
TNFa (human, natural, ultrapure grade, specific activity lo7
unitshg of protein) was carried out at different concentrations
in serum-free DMEM for various durations up to 48 hours.
Both cytokines were from Calbiochem (San Diego, CA).
After cytokine treatment, the culture media were
collected and the cells were detached with Earle's solution
containing 5 mM EDTA. Aliquots of the cell suspensions
were used for cell counting and for evaluation of cell
viability. Cell viability, evaluated after cytokine treatment,
was always >99%, by trypan blue exclusion, and was not
affected by the cytokine treatment. The cell suspensions
were centrifuged and the cell pellets were lysed by ultrasonication in 0.25M sucrose (0.2 ml per lo6 cells).
Cysteine proteinase activity measurement. Cysteine
proteinase activity and latent cysteine proteinase activity were
determined enzymatically, at 37"C, with the fluorogenic substrate Z-Phe-Arg-AMC (Bachem, Bubendorf, Switzerland),
using a Cobas Fara I1 centrifuge analyzer (Hoffmann-La
Roche, Basel, Switzerland). Latent cysteine proteinase activity
was activated by limited proteolysis using pepsin (Sigma, St.
Louis, MO), as described by Qian et a1 (25). The apparatus
distributed successively the following: (a) 12.5 pl of sample,
214 pl of 50 mM MES buffer, pH 5.2, containing 1.8 mM
dithiothreitol and 1 mM EDTA, and 12.5 pl of the pepsin
solution (0.9 mg/ml of pepsin in 0.2M sodium acetate, pH 4.2)
or 12.5 pl of the pH 4.2 buffer alone; (b) after 5 minutes, 12.5
pl of 2.5 p M aminomethyl coumarin used as an internal
standard; (c) after 14 minutes, 31 pl of 0.25 mM Z-Phe-ArgAMC; and (d) after 5 minutes, 20 pl of 33 pM E-64. Fluorescence was recorded at each step. The reaction was linear
throughout the assay.
Cysteine proteinase activity was calculated as the
difference between the fluorescence measured with and
without E-64 (26), and was expressed as unitshter (1 unit of
activity corresponds to the release of 1 pmole of aminomethyl coumarinhninute).
Ion-exchange high-performance liquid chromatography (HPLC). Ion-exchange HPLC (IE-HPLC) was carried
out using a CX-300 column (4.6 x 30 mm; Touzart et
Matignon, Cleveland, OH). The system was equilibrated in
0.01M potassium phosphate buffer, pH 5.8, at a flow rate of
0.4 mYminute (27). Samples of culture medium (0.2 ml)
derived from stimulated or unstimulated cells were injected.
Elution was carried out with a gradient of KCI (O-lM) in the
same buffer (27). Eluates were collected in 0.4 ml-fractions,
and the enzymatic analyses were performed as described
above, using 1 2 . 5 ~ 1samples. Standards of purified cathepsin B and L (Calbiochem) were chromatographed under the
same conditions.
Immunoblotting with anti-human cathepsin B antiserum. Cultured cells from RA and OA patients were analyzed for intracellular and extracellular cathepsin B by
HUET ET AL
774
~
A0
B
0
200
400
600
M O
imo
800
low
TNF a ( W d )
2w
400
64)
TNF a (U/ml)
Figure 1. Effect of tumor necrosis factor a (TNFa) stimulation on
cysteine proteinase activity after pepsin hydrolysis in medium from
culture of synovial fibroblast-like cells from patients with A, osteoarthritis and B, rheumatoid arthritis. Cells were cultured for 48
hours in the presence and absence of various concentrations of
TNFa. and cysteine proteinase activity was analyzed using the
fluorogenic substrate 2-Phe-Arg-AMC. Activity is reported as the
difference between fluorescence with and without E-64 (see Patients
and Methods for details).
lysis, both in culture media and cell lysates of synovial
fibroblast-like cells from synovial tissues of 3 patients
with RA and 3 patients with OA. Spontaneous production of cysteine proteinase activity in culture medium
after pepsin proteolysis was similar in patients with
OA (l,OOO-3,000mU/liter) and with RA (2,000-2,300
mU/liter). We then tested the effect of various cytokines on synovial cell secretion of cathepsin activity.
The addition of TNFa to the culture medium
led to an elevation of latent cysteine proteinase activity, both in cells from patients with OA (Figure IA)
and in cells from patients with RA (Figure 1B). The
stimulating effect of TNFa appeared maximal among
the various patients' cells at concentrations of 10&500
units/ml. TNFa increased the secretion of cathepsin
activity in synovial fibroblast-like cells by 9.3-fold,
3.1-fold, and 1.5-fold in the 3 OA patients and by
2.7-fold, 3-fold, and 2.5-fold in the 3 RA patients,
respectively. Basal cysteine proteinase activity in the
absence of pepsin treatment was very low and did not
change after treatment with TNFa. There was only a
transient increase in cathepsin activity of cell lysates
after treatment with TNFa, with peak levels reached
after 8 hours of treatment (data not shown).
The effect of treatment with IL-1 on secreted
immunoblotting. Samples of culture medium and cell lysate
were analyzed after migration on a 5-30% polyacrylamide
gradient gel under reducing or nonreducing conditions (28).
The proteins were transferred to a nitrocellulose membrane
according to the method described by Vaessen et a1 (29). The
membrane was then treated with sheep anti-human cathepsin B
antiserum (ICN Immunobiologicals, Lisle, IL) diluted 200-fold.
Antigen-antibody complexes were detected with peroxidaseconjugated anti-sheep Ig antibody (ICN Biomedicals, Costa
Mesa, CA) treated with 4-chloro-I-naphthol (Sigma).
Immunocytochemical testing. Synovial fibroblast-like
cells were grown in glass culture chamber slides (Poly Labo,
Strasbourg, France). The effect of TNFa was tested at a
concentration of 500 unitslml in serum-free DMEM for 24
hours. Cathepsin B was immunolocalized in cultured cells.
After fixation by 80% ethanol for 5 minutes, sheep antihuman cathepsin B antibody followed by avidin-biotinperoxidase-conjugated anti-sheep Ig antibody (Amersham,
Buckinghamshire, UK) was applied.
RESULTS
Effects Of IL-' and TNFa On 'ysteine proteinase
activity. Cysteine proteinase activity was measured
with and without previous activation by pepsin proteo-
Bo
2
4
6
IL-1
8
10
(Uml)
Figure 2. Effect of interleukin-1 (IL-1) stimulation on cysteine
proteinase activity after pepsin hydrolysis in medium from culture of
osteoarthritis (A)
fibroblast. . and rheumatoid arthritis (B)
. . svnovial
.
like cells. See Figure 1 for details of culture.
TNFa AND IL-1 STIMULATION OF CYSTEINE PROTEINASE
775
Table 1. Stimulation of cysteine proteinase activity secreted by
synovial fibroblast-like cells from OA and RA patients, after treatment with T N F a or IL-I*
Factor by which cysteine proteinase
activity was increased
~~
Cell
source
OAl
0A2
OA3
RA 1
RA2
RA3
After TNFa
treatment
After IL-1
treatment
9.3
3.1
12.0
1.5
1.5
-
2.7
3.0
2.5
2.5
1.5
-
* OA = osteoarthritis; RA = rheumatoid arthritis; TNFa = tumor
necrosis factor a ; IL-I = interleukin-I.
cathepsin activity was also tested in 4 cases. IL-1
stimulated latent cysteine proteinase activity released
into the culture medium. The maximal increase in
activity was 12-fold and 1.5-fold in the 2 OA patients,
and 2.5-fold and 1.5-fold in the 2 RA patients (Figures
2A and B). The results shown in Figure 2 were
obtained with synovial fibroblast-like cells derived
from the same patients as those for TNFa illustrated in
Figure 1, and these results are summarized in Table 1 .
Generally, the response to TNFa was somewhat
higher than the response to IL-1, and we therefore
characterized this response in more detail.
IE-HPLC elution profiles. The elution profile of
the latent cysteine proteinase activity released by
synovial fibroblast-like cells was compared with the
profiles of standard human cathepsin B and L , by
IE-HPLC. Samples of medium from cultures of synovial fibroblast-like cells from an RA and an OA patient
were evaluated, with and without stimulation by
TNFa. The purified cathepsin B standard eluted at a
retention time of 17 minutes, and the purified cathepsin L standard eluted at two peaks: one with a retention time of 18 minutes, and the other, larger peak at 22
minutes (Figure 3A).
The cysteine proteinase activity after pepsin
treatment was quite low in the media from unstimulated cells of both RA and OA patients. Activity in the
media from the OA patient’s synovial fibroblast-like
cells eluted in a complex pattern of peaks with retention times of 16-23 minutes, which possibly suggests
the presence of both cathepsins L and B. Activity in
the media from the RA patient’s cells eluted at retention times of 16-19 minutes, which is consistent with
cathepsin L, cathepsin B, or both (Figures 3B and C).
After stimulation by TNFa, there were large increases
in the activity eluted in the peak at 17 minutes, both in
A
+
retention t i m (min)
CmIhqinL
+ ulhcuinB
r
-
retention time (min)
rlhDlllNFc
+ *lNFm
Figure 3. Ion-exchange high-performance liquid chromatography
of A, purified cathepsins B and L, B, culture media from osteoarthritis synovial fibroblast-like cells and C, rheumatoid arthritis
synovial fibroblast-like cells, either unstimulated or stimulated with
tumor necrosis factor a (TNFa). Cysteine proteinase activity was
measured after pepsin hydrolysis in the fractions collected after
chromatography. See Patients and Methods for details.
the OA (Figure 3B) and the RA (Figure 3C) culture
supernatants. The retention times of cathepsin activity
in the supernatants after TNFa stimulation were the
same as that of the cathepsin B standard, which
strongly suggests that TNFa substantially augments
the level of cathepsin B secreted by these cells. A
HUET ET AL
776
portion of the activity also eluted at the column
exclusion point, particularly for the RA supernatants.
This possibly suggests that additional, uncharacterized
cathepsin activity is also secreted by RA synovial
fibroblast-like cells after TNFa stimulation.
Results of immunoblotting studies of cathepsin B.
Intracellular and extracellular cathepsin B from cultured cells obtained from OA and RA synovial tissues
were investigated by immunoblotting using antihuman cathepsin B antiserum (Figure 4). Samples of
culture media and cell lysates were analyzed with and
without 2-mercaptoethanol reduction and were compared with standard human cathepsin B. Standard
human cathepsin B was visualized as a band of M , 30
without 2-mercaptoethanol treatment and as a band of
M , 26 after treatment. Although it is difficult to distinguish the size difference between the reduced and
nonreduced forms on the separate gels, a change in
molecular weight was clearly seen when cathepsin B
(reduced and nonreduced) was run on the same gel
(data not shown).
The intracellular form of cathepsin B was the
same molecular weight in cells from both the OA and
the RA patients, appearing as a major band of M, 30
under nonreducing or reducing conditions. The extracellular form(s) of cathepsin B was also the same
molecular weight in both patients, appearing as a band
of M , 43 under nonreducing conditions, and as a major
band of M, 43 with 2 minor bands ( M , 35 and 30) under
reducing conditions,
Immunocytochemistry. The stimulating effect of
TNFa in serum-free medium was also analyzed by
immunocytochemistry. In the absence of added cytokine, the synovial fibroblast-like cells had long, slender cytoplasmic processes and appeared unstained
(Figure SA). After stimulation by TNFa, at a concentration of 500 units/ml for 24 hours, some of the cells
stained with small dark-brown spots, which were
scattered at the periphery of the cytoplasm (Figure 5B).
DISCUSSION
Our results show that TNFa and IL-1 stimulate
the secretion of latent cysteine proteinase activity by
cultured synovial fibroblast-like cells from explants
isolated from patients with RA and patients with OA.
The effects of the cytokines were dose-dependent ;
however, the level of stimulation varied between 1.5fold and 12-fold, according to the explant and the
cytokine tested. No correlation between either the
basal level of secretion or the level of TNFa or IL-1
Figure 4. Immunoblotting with anti-human cathepsin B antiserum
under A, nonreducing and B, reducing conditions. Purified cathepsin
B (lane I), synovial fibroblast-like cell supernatants (lanes 2 and 4),
cell lysates (lanes 3 and 9, or molecular weight markers (lane 6)
were separated by polyacrylamide gel electrophoresis and blotted
onto nitrocellulose. Cathepsin B was identified with sheep antihuman cathepsin B antiserum followed by peroxidase-conjugated
anti-sheep antibody. Lanes 2 and 3 are synovial fibroblast-like cell
supernatants and lysates from a patient with osteoarthritis, lanes 4
and 5 from a patient with rheumatoid arthritis.
stimulation and the underlying arthritis was identified.
Apparently, after several passages, the type of arthritis did not affect the responsiveness of the cells to
TNFa and IL-1 stimulation; this feature has also been
reported for the response to PDGF (19). In our exper-
TNFa AND IL-1 STIMULATION OF CYSTEINE PROTEINASE
777
Figure 5. Cathepsin B immunostaining of rheumatoid arthritis synovial fibroblast-like cells. Cultured cells were grown on slides and
treated with serum-free Dulbecco’s modified Eagle’s medium, for 24
hours, in the absence (A) or presence (B) of tumor necrosis factor a.
The cells were then fixed with 80% ethanol and treated with sheep
anti-human cathepsin B and avidin-biotin-peroxidase-conjugated
anti-sheep Ig.
iments, stimulation was somewhat greater with TNFa
than with IL-1.
The latent cysteine proteinase activity secreted
by synovial fibroblast-like cells was activated by pepsin hydrolysis. In unstimulated synovial fibroblast-like
cells, the relatively low basal activity eluted in IEHPLC at the same retention times as lysosomal cathepsins B and L. After TNFa treatment, the activity
eluted at the same retention time as cathepsin B,
which suggests that cathepsin B is dominant after
TNFa stimulation. The posttranslational processing of
human cathepsin B is a multistep sequential process of
signal peptide, propeptide, and light-heavy chain
cleavage events (30). In cultured human dermal fibroblasts, cathepsin B is synthesized as an N-glycancontaining precursor of M , 44.5, which is in part
converted to a form of M , 46 (31). These forms
apparently contain the same peptide structure as the
secreted 43-kd form. Cleavage of the 62-amino acid
propeptide results in a form with M , 33, which can
then be further cleaved into M , 27 and 6 forms (31).
This latter heav y-light chain cleavage of cathepsin B
converts the protein from a single-chain form to a
2-chain form, and varies according to the cell type
(30). Treatment of fibroblast cultures with leupeptin
results in an accumulation of the 33-kd form and an
increase in cathepsin B activity, which suggests that
the 33-kd form is the most active form in cultured skin
fibroblasts (31). By immunoblotting, we found that
cathepsin B was present in both synovial fibroblastlike cell supernatants and cell lysates.
In the culture medium, cathepsin B was detected as a major band of M , 43. This corresponds to
the size of the precursor form of cathepsin B prior to
cleavage of the propeptide (32), and is the form of
cathepsin B that has generally been reported to be
secreted by other cell types. In the cell lysates, cathepsin B was detected at a molecular weight of
778
approximately 30 kd. This band likely corresponds to
the single-chain enzyme prior to cleavage. The purified
enzyme showed lower molecular weight after reduction, which suggests that the purified enzyme is in the
2-chain form and loses the 6-kd light chain after
reduction. The intracellular synovial fibroblast-like
cell form showed no change in molecular weight after
cleavage, which suggests that it remained as a single
chain. It is possible that the absence of the 6-kd light
chain in the reduced purified control protein was the
result of inadequate resolution of this size protein in
the gel we used or nonreactivity of the antibody with
this portion of the protein.
Immunocytochemistry showed that TNFa increased levels of cathepsin B in cultured synovial
fibroblast-like cells. These results suggest that increased secretion results, at least in part, from increased intracellular accumulation of cathepsin B,
which is consistent with our data showing a transient
increase in intracellular cathepsin B levels after stimulation with TNFa (data not shown). The synthesis or
secretion of molecular forms of cathepsin B may be
modified by malignant transformation or dedifferentiation. Latent forms of cathepsin B have been found in
ascitic fluid from cancer patients, in culture medium of
ascites cells from cancer patients (33,34), and in culture medium of rabbit skin fibroblasts and rabbit V2
carcinoma cells (35). The V2 form showed unusual
stability at pH >7.0 (35). We have not yet characterized the pH-related stability of the cathepsin B secreted by synovial cells. However, its activity in the
latent form was apparently stable in culture media at a
pH of -7.4, since it had enzymatic activity after
pepsin activation. Dedifferentiated chondrocytes cultured in monolayer produce and secrete a considerable
amount of cathepsin B of a 34-kd form, which also
shows a high resistance to denaturation at neutral
alkaline pH (36).
TNFa, a monocytelmacrophage-derived cytokine, showed a stimulating effect on the secretion of a
latent cysteine proteinase activity by synovial fibroblast-like cells. This activity appeared to be primarily
cathepsin B. IL-1 also stimulated cathepsin B secretion in 4 of the 6 cases studied. A similarity in the
biologic effects of IL-1and TNFa has frequently been
described. Both cytokines are able to induce a cytotoxic effect on certain transformed cells and a stimulating effect on collagenase and PGE, production by
synovial cells, as well as inducing bone resorption
(37). Both cytokines are considered important mediators in the pathogenesis of cartilage degradation and
HUET ET AL
are frequently found in the synovial fluid of RA and
OA patients (22-24). TNFa has been immunolocalized
to the cartilagepannus junction in RA (38). The
induction of arthritis in transgenic mice expressing
human TNFa (39) has further implicated this cytokine
in the pathogenesis of RA. The potential role of
cathepsin B in the degradation of articular connective
tissues, however, has been shown by Trabandt et al,
who demonstrated (a) a high level of cathepsin B
messenger RNA in RA synovial cells and (b) the
immunolocalization of cathepsin B to synovial tissue
cells at sites of cartilage and bone destruction (40).
Thornberry et al (41) have recently described a
novel heterodimeric cysteine proteinase responsible
for the activation of the IL-1p precursor (pIL-1p) to
mature IL-1p (mIL-1p) in monocytes. This enzyme
(interleukin- 1wonverting enzyme, or ICE) does not
show sequence homology with any known cellular
cysteine proteinases. ICE appears to be a pIL-1&
specific processing enzyme, which cleaves two Asp-X
bonds in the pIL-lp sequence, Asp 116-Ala 117 and
Asp 27-Gly 28. The minimal best substrate for the
enzyme was the peptide Ac-Tyr-Val-Ala-Asp-NHCH,, with broad substitution tolerated in the P2 position. Cathepsin B, in contrast, is a very active proteinase involved in intracellular protein turnover and has a
much broader substrate specificity (27). It is possible,
however, that interactions between these and/or other
proteinases of the cathepsin family might have a role in
the activation of IL-1, although apparently IL-1 activation is primarily a cell-associated event occurring in
the monocyte.
Our results showing a stimulating effect of
TNFa on secretion of latent cysteine proteinase activity by synovial fibroblast-like cells suggest that this
cytokine plays an important role in the mediation of
cartilagelbone degradation in RA through the secretion
of a cathepsin B precursor. Further studies may help
to determine more precisely how cathepsin B is activated in the joint tissue, whether it maintains its
stability after activation, and whether its secretion can
be modulated by other biologically active molecules
such as transforming growth factor p and retinoic acid,
which can inhibit metalloproteinase secretion.
ACKNOWLEDGMENTS
We thank Professors C. Fontaine and H. Mestdagh
for providing samples of synovial tissue.
TNFa AND IL-1 STIMULATION OF CYSTEINE PROTEINASE
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