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Extracellular presence of the lysosomal proteinase cathepsin B in rheumatoid synovium and its activity at neutral pH.

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EXTRACELLULAR PRESENCE OF THE LYSOSOMAL
PROTEINASE CATHEPSIN B IN RHEUMATOID
SYNOVIUM AND ITS ACTIVITY AT NEUTRAL pH
JOHN S. MORT, ANNELIESE D. RECKLIES, and A . ROBIN POOLE
The presence of the lysosomal proteinases cathepsin B and cathepsin D at extracellular sites in
rheumatoid synovium was demonstrated using the antibody capture technique. Unlike cathepsin D, the cysteine proteinase cathepsin B was commonly detected only
at the edges of the synovial explants. Radioimmunoassay
and enzyme activity assay of these proteinases demonstrated that both were released from rheumatoid synovia1 cells in comparable amounts. Since lysosomal cathepsin B is unstable and denatured at physiologic pH and
the antibody used only recognizes inactivated enzyme,
we believe the selective detection of cathepsin B at the
edge of the synovium may be due to the proteinase
maintaining a native conformation within the explant,
where the pH may be low enough to permit this. By use
of a fluorescent substrate in a sensitive, continuous
enzyme assay, cathepsin B was shown to express significant activity at neutral and alkaline pH before being
inactivated. This and earlier work from this laboratory
indicate that cathepsin B secreted by rheumatoid synovial cells may possess extracellular activity in vivo and be
~From the Joint Diseases Laboratory, Shriners Hospital for
Crippled Children, Montreal, Quebec, Canada and the Department
of Surgery, McGill University, Montreal, Quebec, Canada.
Supported by research grants from the Shriners of North
America and the National Cancer Institute of Canada.
John S . Mort. PhD: Assistant Professor of Experimental
Surgery. McGill University: Anneliese D. Recklies. PhD: Assistant
Professor of Experimental Surgery, McGill University: A. Robin
Poole, PhD: Professor of Experimental Surgery, McGill Univcrsity
and Director, Joint Diseases Laboratory.
Address reprint requests to Dr. J . S. Mort, Joint Diseases
Laboratory, Shriners Hospital for Crippled Children. 1529 Cedar
Avenue, Montreal, Quebec, Canada H3G 1A6.
Submitted for publication August IS, 1983; accepted in
revised form January 4. 1984.
Arthritis and Rheumatism, Vol. 27, No. 5 (May 1984)
involved in the degradation of connective tissue macromolecules.
Destruction of articular tissue such as cartilage
is a lasting consequence of rheumatoid arthritis, and
leads to loss of joint function. Proteinases released
from polymorphonuclear leukocytes in the synovial
fluid, the inflamed synovium, and invading pannus are
important mediators of the degradation of collagen and
proteoglycan, which largely constitute the matrix of
articular cartilage (1). A specific group of these enzymes is the group of lysosomal proteinases, such as
cathepsin D and cathepsin B, which when released
extracellularly have a high destructive potential, although they are usually not considered to be active at
neutral pH.
Earlier, the extracellular presence of the lysosoma1 aspartic proteinase cathepsin D was demonstrated in rheumatoid synovium (2) using the in vitro
enzyme capture method (3). With this technique rheumatoid synovial explants are maintained in organ
culture in the presence of antiserum to the proteinase
under study. Enzyme released from the cells is then
captured locally by antibodies, with the formation of
minute extracellular immune complexes which can be
subsequently detected in tissue sections by using a
fluorescent second step antiimmunoglobulin antibody.
In contrast to rheumatoid synovium, cathepsin D was
never detected in extracellular sites in noninflamed
synovium from patients with traumatized meniscoid
cartilage (2).
In the present study we have extended this
approach to the investigation of the extracellular re-
MORT ET AL
510
cathepsin B serum, or sheep anti-human cathepsin D serum
(all 10%). Before use, these sera were acid- and heat-treated
(11). After 48 hours of culture in the presence of immune
serum, the tissue fragments attached to the Millipore membrane were placed in 7.0% gelatin in phosphate buffered
saline (PBS) and frozen in liquid nitrogen (2).
For penetration studies, explants were cultured in
fetal calf serum as above, then incubated with medium
containing 10% normal sheep serum (heat- and acid-treated)
with or without 30 $3 of '2'I-cathepsin B. After 6 hours the
tissues were frozen as above. For enzyme secretion studies,
explants were cultured as above in RPMI medium containing
10% heat-inactivated fetal calf serum. Medium was collected
on days 1 and 3, and cathepsin B protein content was
determined by radioimmunoassay. DNA content was determined in the explants at the end of the culture as described
previously (12). Culture in sheep serum (as used for enzyme
capture) was not possible due to the constraints of the
radioimmunoassay for cathepsin B, which utilizes a sheep
antiserum (8).
PATIENTS AND METHODS
Localization of cathepsins B and D. Frozen tissue
was cryostat-sectioned (4 pm). Sections were washed for 15
minutes in PBS, fixed in 4% freshly prepared formaldehyde
Materials. Cathepsins B and D from human liver
(2) for 2 minutes, then washed for an additional 30 minutes in
were purified, and specific polyclonal hyperimmune sheep
PBS. The sections were stained with fluoresein-labeled
antisera containing IgG antibodies were prepared and specirabbit anti-sheep IgG, diluted at 1/10 with PBS, for 30
ficities demonstrated as prevously described (6-8). Rabbit
minutes, and then washed in PBS as above. Cell cytoplasms
anti-sheep IgG, fluorescein-labeled, was purchased from
were counterstained with eriochrome black (1150 dilution in
Dako immunoglobulins (supplied by Cedarlane LaboraPBS) and viewed for green and red fluorescence with a Zeiss
tories, Hornby, Canada). CBz-Phe-Arg-4-methyl-7-couma- Photomicroscope 111 (13). Sections from tissue cultured with
rylamide was from Bachem (Bubendorf, Switzerland) and
or without '251-labeledcathepsin B were placed onto gelatinBz-Arg-pnaphth lamide was from Sigma (St. Louis, MO).
coated glass slides and then dipped in Ilford Nuclear ReCarrier-free Na I 'I was from New England Nuclear (Monsearch Emulsion gel and stored frozen at -20°C. After 2
treal, Canada). Eriochrome black was supplied by Difco
months, they were developed using Kodak D170 developer
Laboratories (supplied through BDH, Montreal, Canada).
and counterstained with 0.1% toluidine blue in 5% ethanol in
Iodination of cathepsin B and radioimmunoassay.
water.
Cathepsin B was '*'I-labeled by the chloramine T method (9)
Proteinase assays. For routine assay, cathepsin B
and separated from free '"I by Sephadex G25 chromatograactivity in culture medium was measured using Bz-Arg-Pphy. The radioimmunoassay for cathepsin B was as previnaphthylamide with a 16-hour incubation as previously deously described (8).
scribed (12,14). Cathepsin D activity was measured using
Patients and synovia. All 5 patients studied had clasacid-denatured hemoglobin as the substrate (1 5).
sic or definite rheumatoid arthritis according to the AmeriFor pH activity profile measurement of cathepsin B,
can Rheumatism Association criteria (10). All patients had
the fluorimetric substrate CBz-Phe-Arg-4-methyl-7-coumarbeen receiving antiinflammatory drugs for at least 1 month
ylamide (5 pM) was used (16). Incubations contained 50 mM
preceding the time of surgery. These drugs included steroids
sodium PIPES (adjusted to the required pH), 1 mM EDTA,
and nonsteroids such as salicylates, gold, and chloroquine.
and 2 mM cysteine. Human liver cathepsin B was diluted
At the time of surgery, patients exhibited active disease.
into 20 mM sodium MES, pH 6.0, 1 mM EDI'A, 2 mM
Synovia all appeared macroscopically inflamed, and microcysteine, 0.1 mglml bovine serum albumin. The incubation
scopically were hypercellular, with villi and extensive monomedium (0.95 ml) without enzyme was preincubated to 37°C
nuclear cell infiltration.
and the reaction started by the addition of purified cathepsin
Culture of synovial explants for detection of extracelluB (33 ng in 0.05 ml). The increase in fluorescence was
lar proteinases. Rheumatoid synovia removed during 5 separecorded continuously (excitation at 350 nm, emission at 460
rate joint replacements were each cut into pieces approxinm) for 4-6 minutes and the initial slope of the reaction was
mately 2 X 4 mm, placed on Millipore filters (3 p,m pore
determined. At the upper pH values studied, decrease of
size), and each was cultured on expanded metal grids in 1.5
activity with time of assay was significant. This loss of
ml RPMI 1640, (Gibco, Grand Island, NY) containing 10%
activity followed a pattern of simple exponential decay from
fetal calf serum (previously heated at 56°C for 30 minutes) in
which the half-life of cathepsin B at pH 7.5 and pH 8 was
a 95% air, 5% C 0 2 atmosphere. After 24 hours the medium
calculated. No change in decay rate was seen over a %-fold
was replaced and the tissue cultured as above for an addirange of enzyme concentration (2.6-130 nglml), indicating
tional 36 hours. The medium was then replaced with RPMI
that this inactivation is a property inherent to the enzyme
mcdium containing normal sheep serum, sheep anti-human
and not due to intermolecular self-digestion.
lease of the cysteine (thiol) proteinase cathepsin B
from rheumatoid synovia, and we have compared its
release with that of cathepsin D. Cathepsin B is
capable of degrading various connective tissue components such as proteoglycan and collagen (4) and fibronectin (5). If released into the extracellular matrix, it
could be an important agent in connective tissue
destruction such as that seen in rheumatoid arthritis.
We present data which indicate that both cathepsin D
and cathepsin B are secreted from rheumatoid synovial cells, and show that for limited periods, extracellular cathepsin B could survive in its native form in
synovium and express activity.
Y
CATHEPSIN B IN RHEUMATOID SYNOVIUM
51 1
Table 1. Secretion of cathepsin B and cathepsin D-from cultured
synovial explants*
Day
1
3
Cathepsin B
(ndkg DNA
2.5
2.1
?
?
0.2
0.1
Cathepsin D
M TYr
Eq/16 hours/
pg DNA
10.1 2 3.2
3.3 2 0.3
* Cathepsin B release from the synovium of 1 rheumatoid arthritis
patient was measured by radioimmunoassay. and cathepsin D by
activity against acid-denatured hemoglobin. Data are mean ? SD
from 6 explants.
RESULTS
Detection of cathepsins B and D in culture medium of rheumatoid synovial explants. Explants of rheumatoid synovium were maintained in organ culture for
Figure 2. Nonimmune sheep serum control. No particulate immune complex staining is seen. A large positive area is seen in the
upper left of the figure. This is an example of large endogenous
immunoglobulin deposits sometimes seen in both test and control
sections. The section was counterstained using eriochrome black
(bar = 10 p n ) .
Figure 1. Extracellular localization of cathepsin D in rheumatoid
synovium by the enzyme capture method. Immune complexes
containing cathepsin D appear as intensely fluorescent points which
occur throughout the full extent of the explant. The area shown is
from well below the natural synovial surface. The section was
counterstained using eriochrome black. to show cytoplasmic contents (bar = 10 Km).
up to 3 days, with the medium replaced at day 1.
Cathepsin D activity was clearly detectable (Table 1).
Radioimmunoassay of cathepsin B showed that the
cysteine proteinase was also released into the culture
medium (Table I). In marked contrast, assay with
synthetic substrate showed no cathepsin B activity.
This finding is not surprising since lysosomal cathepsin B is unstable under the conditions used for culture
(pH 7.3, 37"C), in which it is rapidly and irreversibly
denatured (14,17). In fact, the antiserum used in the
radioimmunoassay recognizes only denatured cathepsin B (6,9), and the enzyme must be denatured to
render it reactive with the antibody.
The accumulation of both enzymes was reduced by about 40-60% when explants were cultured
in the Presence of the Protein synthesis inhibitor
cycloheximide (2 pg/ml), indicating that active secre-
MORT ET AL
512
Figure 3. Extracellular localization of cathepsin B in rheumatoid synovium by the enzyme capture method.
Immune complexes containing cathepsin B appear as intensely fluorescent points at the natural edge of the tissue.
The section was counterstained using eriochrome black (bar = 10 Wm).
tion was occurring. Values for cathepsin B represent
total enzyme protein detectable by radioimmunoassay, but since cathepsin D was determined
by its activity using acid denatured hemoglobin as
substrate, cathepsin B and D concentrations cannot be
directly compared. However, using specific activities
for human cathepsin D as reported by Barrett (IS), the
observed activities are equivalent to approximately 210 ng cathepsin D/pg tissue DNA. These values fall
into the same protein concentration range as those
observed for cathepsin B release.
Extracellular localization of cathepsins B and D
in synovial explants. By maintaining rheumatoid synovial explants in organ culture, in the presence of
antiserum to cathepsin D, the extracellular presence of
this lysosomal proteinase throughout most synovial
explants was demonstrated as previously described
(2). The extracellular localization of immune complexes formed from the reaction of antibody with secreted
proteinase was clearly demonstrated by counterstaining cell cytoplasms with the red fluorescent dye
eriochrome black. Cells fluoresce red, in sharp contrast to the extracellular immune complexes, which
fluoresce green on staining with fluorescein-labeled
rabbit antibody to sheep immunoglobulin (2,3). The
tissues were deliberately cultured first in the absence
of antibody to permit proteinases to be released from
dead or dying cells, such as at the cut edges of the
explants. As before (2), reaction of antibody with such
damaged cells resulted in intense ccllular staining.
This was rarely observed following this pre-culture
period.
Extracellular immune complexes of cathepsin
D and specific antibody were localized in frozen
sections as discrete, rounded, microparticulate staining throughout the explants (Figure I). These complexes were observed in all 3 patients examined.
Although they were sometimes more commonly seen
at the natural edges of explants, they were always
observed throughout the synovial explants. Control
explants cultured in the presence of normal sheep
serum showed no such discrete particulate immune
staining (Figure 2), although occasional larger, more
irregular, less intense particulate extracellular staining
was seen, possibly representing immune complexes
already present in the tissue. These were also detected
without culture (not shown).
Explants from 4 patients, including the 3 noted
CATHEPSIN B IN RHEUMATOID SYNOVIUM
above, were cultured in the presence of antiserum to
cathepsin B. They showed finer particulate extraccllular staining similar to that seen for cathepsin D, but in
this case, it was exclusively localized at or close to the
edges of the tissue (no more than 4-5 cells deep) and
was particularly common at natural non-cut surfaces.
In every patient, staining was relatively absent from
the interiors of these explants under this outer lining
cell layer (Figure 3 ) .
We also showed that extracellular cathepsin B
can easily penetrate throughout the synovial matrix,
by demonstrating the even distribution of grains in
autoradiographs of tissue cultured in the presence of
"'I-labeled cathepsin B (Figure 4). These grains were
always selectively concentrated over the tissue being
examined. Since cathepsin B is likely to be released by
synovial cells throughout the tissue and the enzyme is
513
5 10
7.0
610
8 :O
PH
Figure 5. Activity (pH) profile of purified human liver cathepsin B.
showing the initial activities at each pH value.
Figure 4. Autoradiographic visualization of '"I-labeled cathepsin
B incubated with rheumatoid synovium in organ culture. The natural
edge of the tissue is shown in the upper right corner. Grains duc to
labeled enzyme are present throughout the full extent of the tissue.
The section was counterstained with toluidine blue (bar = 20 pm).
freely diffusible, it seems that while enzyme at the
edge of the tissue must be in a denatured form to be
recognized by the antiserum, any enzyme within the
explant is not denatured and therefore not precipitated
by the antiserum.
Effect of pH on cathepsin B activity. It is often
assumed that lysosomal proteinases cannot be cffective extracellular degradative agents because the pH at
which they have optimal activity is below that expected in the extracellular environment. In the case of
cathepsin B , measurements of the optimal pH of the
enzyme are usually carried out using assays in which
the proteinase must be incubated with the substrate for
a fixed time. If the enzyme is unstable under these
assay conditions, a biased estimate of the true pH
optimum will be obtained by these methods. The
fluorescent substrate CBz-Phe-Arg-4-methyl-7-coumarylamide can be used in a very sensitive continuous
assay which allows the measurement of the initial
reaction rate. When the action of cathepsin B on this
substrate at various pH values was investigated, high
initial enzyme activity was seen up to pH 8 (Figure 5 ) .
The half-lives of the enzyme at pH 7.5 and 8 were
calculated from progress curves and were 7 minutes
and 1.7 minutes, respectively. Below these values the
enzyme remains active for much longer periods (14).
Clearly, therefore, when released from the cell this
lysosomal enzyme has potent, if somewhat shortlived, destructive potential.
MORT ET AL
5 14
DISCUSSION
It is generally understood that lysosomal proteinases display significant activity only at acidic pH
and are thus not thought to cause extracellular tissue
destruction as seen in rheumatoid arthritis. While this
may be so for the aspartic proteinase cathepsin D (19),
our results clearly show that the purified cysteine
proteinase cathepsin B is enzymically active over a
wide pH range and, in fact, shows a similar initial
velocity profile to that of the well-studied plant cysteine proteinase papain (20). With increasing pH, cathepsin B is increasingly unstable, but our studies
suggest that when released into an extracellular milieu
maintained between pH 7-7.5, activity can persist for
a limited period and could cause proteolysis before the
enzyme is denatured. Recent work on human inflamed
gingiva has revealed the presence of cathepsin B-like
cysteine proteinase activity, measurable between pH
7-8, which survives in crevicular fluid (21).
In this comparative study of the release of
cathepsins B and D from synovial cells, we decided to
study rheumatoid synovium since our previous immunohistochemical work failed to demonstrate any
extracellular cathepsin D in noninflamed synovium
from patients at meniscectomy (2). The present study
reveals that when the samc method is used, both the
lysosomal proteinases cathepsin D and cathepsin B are
demonstrable in extracellular sites in rheumatoid synovium. Although both cathepsin B and cathepsin D are
secreted into extracellular sites as revealed by radioimmunoassay and enzyme activity assay, cathepsin B,
unlike cathepsin D, was commonly detected only at
the edges of the synovial explants in culture. Since
cathepsin B is unstable at neutral pH and is only
recognized by antibody in its denatured form (6, I7),
antibodies to the enzyme would only detect the denatured enzyme in culture. Cathepsin D, in contrast, is
stable at physiologic pH and is recognized by antibody
in its native form (22). Thus, the observed restricted
localization of extracellular cathepsitl B to the periphery of the synovium suggests that the extracellular pH
within the synovium is too low for the enzyme to be
denatured and hence recognized and reacted with by
antibody.
Since exogenous radiolabeled cathepsin B
(shown here) and immunoglobulin G antibodies (2) are
free to diffuse throughout the explants as indicated by
autoradiography, it is unlikely that there are any
penetration limitations imposed upon either the proteinases or the antibodies. Hence, it would appear
likely that cathepsin B is released by cells throughout
the explant, which is at an acidic pH except at its
edges. It is, of course, possible that inhibitors of this
cysteine proteinase which are present in body fluids
(23,24) could react with the enzyme and prevent its
detection by the antibody. However, there is no
evidence as yet to indicate that this can happen. It is
unlikely in this case, since cathepsin B undergoes a
conformational change when it is denatured, as indicated by the fact that the antibody only reacts with the
inactive enzyme (6). The epitope(s) exposed on the
denatured enzyme are not accessible in the native
form which reacts with inhibitor, and hence would
likely not be masked.
Under inflammatory conditions, the extracellular pH is known to be decreased to as low as 5.0 at the
surface of macrophages, as shown by work with micro
pH electrodes (25). It was clear from our own work
that the inflamed synovium is metabolically very active, since the culture medium becomes acidic within
24 hours (unpublished observation). Thus it is possible
that cathepsin B is secreted into an “acidic” environment which permits it to express proteolytic activity.
Even if the extracellular pH is maintained at neutrality, our studies indicate that cathepsin B is active long
enough to initiate tissue damage.
ACKNOWLEDGMENTS
We wish to thank Drs. R. L. Cruess, W. Rennie, and
the late E. Rogala (Department of Orthopedics, McGill
University) and Dr. D. Cooke (Queen’s University, Kingston, Ontario) for the supply of rheumatoid synovium obtained during joidt replacement surgery; Isabelle Pidoux and
Michele Leduc for excellent technical assistance; and Nora
Shepard for performing the autoradiography.
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