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Enhanced neutral protease activity in proliferating rheumatoid synovial cells.

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919
ENHANCED NEUTRAL PROTEASE ACTIVITY IN
PROLIFERATING RHEUMATOID SYNOVIAL CELLS
VICTOR B. HATCHER, JOANNE P. BORG, MINDY A. LEVITT, and CAROL SMITH
Proteolytic enzymes associated with rheumatoid
synovial cells (RSC) have been implicated in the degradation of articular cartilage. Proteases have been measured in proliferating rheumatoid synovial cells (PRSC), proliferating nonrheumatoid synovial cells (PNSC), and their nonproliferating counterparts (NPRSC and NP-NSC). The P-RSC and P-NSC exhibit enhanced total surface-associated plus secreted neutral
protease activity, as compared with NP-RSC and NPNSC. The P-RSC exhibited significantly higher protease activity in this category compared to P-NSC. The
RSC also secreted higher levels of secreted proteases
alone compared to NSC. The secreted protease activity
alone was not related to the proliferative state of the
cells. Extractable protease activity was measured in
early-passaged and serially-passaged P-RSC, NP-RSC,
P-NSC, and NP-NSC. Extractable cellular protease activity measured at pH ranges from 5.0 through 8.0 was
not significantly different between P-RSC and NP-RSC
or between P-NSC and NP-NSC. The RSC contained
elevated extractable cellular protease activity measured
at pH ranges from 5.0 through 8.0 compared to extracts
from later-passaged cells. The neutral protease activity
in the early-passaged RSC was also higher than that
From the Departments of Biochemistry and Medicine, Albert Einstein College of Medicine, Montefiore Hospital and Medical
Center.
Supported in part by the New York Arthritis Foundation,
NIH grants AG 01732, HL 16387, and AM 15796, the New York
Heart Association, and the Cystic Fibrosis Foundation.
Victor B. Hatcher, PhD: Associate Professor, Department of
Biochemistry and Medicine (recipient of New York Heart Senior Investigator Award); Joanne P. Borg: student, Tufts University, Boston,
(recipient of summer fellowship, New York Arthritis Foundation);
Mindy Levitt: Research Assistant, Montefiore Hospital and Medical
Center, Bronx, New York; Carol Smith: Associate Professor, Department of Medicine.
Address reprint requests to Victor B. Hatcher, PhD, Albert
Einstein College of Medicine, Montefiore Hospital and Medical Center, 11 1 East 210th Street, Bronx, NY 10467.
Submitted for publication August 25, 1980; accepted in revised form January 28, 1981.
Arthritis and Rheumatism, Vol. 24,No. 7 (July 1981)
measured at pH 6.0 or 8.0. In synovial cells cellular protease activity was related to the proliferative state and
the origin of the cells. The P-RSC exhibited the highest
levels of surface associated plus secreted neutral protease activity. The RSC also possessed the highest levels of extractable protease activity compared to NSC.
The biological stimulus and the mechanism responsible for the degradation of cartilage in rheumatoid
arthritis are not clearly understood. Hypertrophy of the
synovial membrane can probably be correlated with articular cartilage destruction. Harris (1) has suggested
that the cartilage is destroyed by enzymes released from
cells of the advancing pannus. Granda et a1 (2) have reported a correlation between lysosomal cathepsin D in
rheumatoid synovia and the amount of damage to articular cartilage. Poo!e et a1 (3) demonstrated the secretion
of cathepsin D in synovial tissue removed from rheumatoid and traumatized joints.
Collagenase has also been extensively investigated in synovial cells from rheumatoid synovium (48). Dayer et a1 (9) have shown that cells which adhere to
tissue culture dishes following proteolytic dispersion of
the rheumatoid synovium secrete large amounts of collagenase and prostaglandin E. The major source of collagenase was large stellate cells with many dendritic
processes (10,ll). Collagenase and prostaglandin production were stimulated by a soluble factor released by
the mononuclear cells (12,13). Monocyte-macrophages
were primarily responsible for the production of the factor (14). Recently, Dayer et a1 (15) observed that adherent rheumatoid synovial cells which possess decreased
collagenase activity following serial passage of the celIs
could be stimulated by a soluble factor released in vitro
from human blood monocyte-macrophages. Increased
levels of the factor were produced by monocyte-macrophages stimulated by aggregated immunoglobulin, Fc
fragment of the immunoglobulin, or concanavalin A.
HATCHER ET AL
920
Neutral proteases have been investigated in synovial lining cells. Hams and Krane (16) isolated an
endopeptidase from RSC which degraded gelatin to
small fragments. Werb and Reynolds (17) have reported
that endocytosis causes increased secretion of neutral
proteinase from rabbit synovial fibroblasts. Werb et a1
(18) have also shown that synovial cell cultures produced plasminogen activator which can generate plasmin from plasminogen. Latent collagenase produced by
rheumatoid synovium can be activated by plasmin.
In this study, surface associated neutral protease
plus secreted neutral protease, secreted neutral protease
alone, and extractable cellular proteases assayed at pH
values from 5.0 through 8.0 have been compared in proliferating versus nonproliferating and early-passage
versus later-passage rheumatoid (RSC)and non-rheumatoid synovial cell (NCS)cultures. Several observations have now been made concerning each of these cell
types.
MATERIALS AND METHODS
Synovial cell culture. Synovial cell cultures were prepared by using a procedure modified from Dayer et a1 (9).
Rheumatoid cell cultures were isolated from samples of synovium obtained at synovectomy from patients with clinically
defined rheumatoid arthritis. Nonrheumatoid cell cultures
were obtained from samples of synovium from patients with
osteoarthritic joint disease who required reconstructive procedures.
Following collection of the specimens in Dulbecco
modified Eagle's medium (DMEM), the lining cell layer was
stripped off and minced into 1-2 mm pieces and placed in
sterile flasks. The minced tissue was incubated for 3 4 hours
on a Bellco rocker platform at 37OC in 50-100 ml of DMEM
containing 1 mg/ml of collagenase (Sigma Type I, Saint
Louis, MO, 125-250 units/ml) and 3-5 mg/100 ml of testicular hyaluronidase (Wyeth, personal supply) supplemented
with 210 units/ml of penicillin and 80 pg/ml of streptomycin
(Pfizer Laboratories, New York, NY). The Bask was rapidly
agitated by hand for 5-10 minutes to break up any remaining
pieces of tissue. The cells were centrifuged for 10 minutes at
500g, and the cell pellet was resuspended in DMEM containing 1Wo newborn calf serum (Gibco) and placed in flasks or in
plastic Petri dishes (Falcon Plastics, Oxnard, CA). After overnight incubation, the floating cells were removed and the adherent cells were washed several times with phosphate buffered saline (PBS) and treated with 1 ml of 0.125% trypsin
(weight/volume) (Gibco) containing 0.004M EDTA for several minutes in order to temove lymphocytes. The cells were
then placed in DMEM containing 20% newborn calf serum.
Primary cultures which were not passaged for longer than 2
weeks were placed in DMEM containing 5% newborn calf
serum. Cultures were carried in DMEM containing 10% newborn calf serum and subcultured at a 1 :2 split ratio using
0.125% trypsin (Gibco), containing 0.004M EDTA.
Cell counts were done in a hemocytometer after
trypsinization. In experiments utilizing nonproliferating cells,
cultures between passage 0-5 were used in which no increase
in the cell counts was observed for at least 3 days following
confluence. In experiments using proliferating cells, synovial
cells between passages 0-5 were used in the rapid growth
phase, usually 48 to 72 hours following subculture.
Surface plus secreted protease activity. The surface
protease determination was based on the measurement of trichloroacetic acid (TCA) soluble radioactive peptides released
from 'H-labeled casein (19,20). The 'H-acetyl-casein assay is
capable of detecting proteolysis produced by 50-100 pg of
trypsin (2 1,22). The 'H-acetyl-casein, which was prepared by
acetylating casein with 'H-acetic anhydride, had a specific activity of 3,840 cpm/pmol, assuming a molecular mass of
121,800 daltons. In order to measure protease activity bound
to the cell surface, plus ptotease secreted into the medium, the
cell cultures grown in 60 mm Falcon petri dishes were washed
6 times with DMEM without serum. The wash was removed
and 300 11 'H-labeled casein (60 pg casein. 2.0 X lo6 cpm)
which was previously dialyzed against serum-free medium
was added to the plates together with fresh serum-free medium (600 1.11).The plates were then incubated for 180 minutes
at 38°C under 7% CO, in room air. The reaction was terminated by the addition of unlabeled 3% casein (100 pl) dissolved in 1M KCl followed by chilled 6% TCA (200 pl). The
mixture was allowed to remain overnight in an ice slurry after
which the samples were centrifuged at 27,ooOg for 40 minutes,
200 pl of supernatant was added to 10 ml of Riafluor (New
England Nuclear), and then the radioactivity was determined.
In the measurement of surface plus secreted protease
activity, plates without cells were used as negative controls.
Control plates without cells were incubated for 25 hours in
DMEM containing 10%newborn calf serum. The plates were
washed 6 times with DMEM without serum. The medium was
removed, and 300 pl 'H-labeled casein (6 pg casein, 2.0 X lo5
cpm) was added to the plates together with serum-free medium (600 pl). The plates were incubated, and the reaction
was terminated as described for the plates containing cells.
The TCA soluble counts measured on plates not containing
cells were substrated from the TCA soluble counts in the cell
cultures grown in Falcon petri dishes. Approximately 2.0% of
the total radioactivity in the 'H-acetyl casein was TCA soluble
following TCA precipitation on the control plates after incubation for 180 minutes. In the plates containing cells from
2.0-6.0% of the total 'H-acetyl casein, radioactivity was TCA
soluble after incubation for 180 minutes. Following each experiment, the cells were trypsinized and counted with a hemocytometer. The viability of the cells was examined by eosin-y
exclusion at the end of the experiment (21). Viable cells were
observed as evaluated by eosin-y exclusion. In some experiments, the cells were trypsinized, washed 3 times, centrifuged,
lysed, and the cell button counted in Riafluor. Less than
0.02% of the total amount of 'H-labeled casein was measured
in the cell button.
Secreted protease activity. In order to determine if
neutral proteolytic activity was secreted during the assay, replicate plates containing serum-free medium (900 pl) were incubated for 180 minutes at 37OC under 7% CO, in room air.
The medium was removed, centrifuged at 21,OOOg for 5 minutes, and an aliquot (100 pl) incubated for 120 minutes at
37°C with 'H-labeled casein (40 pl). The reaction was termi-
NEUTRAL PROTEASE ACTIVITY
nated with unlabeled 3%casein (100 pl) and 6% TCA (200 pl)
under the conditions described above. An aliquot (100 p l ) was
counted for radioactivity.
Extractable cellular protease activity. NSC and RSC
cultures were extracted by freezing and thawing (3 times) in I
ml of 0.05Msodium phosphate buffer, pH 7.5, containing 1M
KCI and 0.1% Triton X-100. After centrifugation at 26,OOOg
for 60 minutes, the precipitate was removed and the supernatant was used for the determination of proteolytic activity.
In the enzyme blank supernatant boiled for 1 minute was
used. A 20-pl aliquot in triplicate of the supernatant was
added to 20 p1 of buffer (1Mphosphate buffer, p H 5.0, 6.0, 7 . 0
or 8.0) in microcentrifuge tubes. The ’H-acetyl-casein (6 pg, 2
x 1CP cpm) was added in a volume of 20 pl, and the incubation was performed for 60 minutes at 4OOC. At the end of
the incubation, 5 0 pl of the unlabeled cold casein (3% w/v in
5 0 m M sodium phosphate buffer, pH 7.0, containing 2M KCI)
and 100 p1 of ice cold 6% TCA (w/v) were added. The contents of the tube were agitated vigorously on a Vortex mixer
and incubated overnight in an ice slurry. Following centrifugation at 26.000g for 45 minutes, the top of each tube was
sliced off and 100 pl of the supernatant was removed, added to
10 ml of Riafluor, and the radioactivity was determined. The
standard Student’s t-test was used for comparison of means
between the different groups (23).
921
Table 1. Surface plus secreted neutral protease activity in
rheumatoid and nonrheumatoid synovial cells
__
Synovial cells.
~
~~~
Passage
number
Protease activity,
units/l05 cellst
4
0
0.4
1.1
2.0
0.8
~.
~
Nonrheumatoid
Nonproliferating
N3
N5
N6
N6
N8
N8
N 12
N 13
0
0
0
Mean f SD
0.5 f 0.7
Proliferating
N3
N7
N8
N9
N 13
Mean SD
Total Mean f SD
*
I
2
4
2
3
I .6
3.6
2.6
2.4
3.1
2.7 f 0.8$
1.4 2 1.3
Rheumatoid
Nonproliferating
RA 4
0.1
RA4
1.8
I .o
RA 5
RA 5
RESULTS
RA6
RA 1 1
Surface plus secreted neutral protease activity.
Proteolytic activity due to proteases associated with the
cell surface, plus that secreted into the medium, were
measured on proliferating rheumatoid synovial cells (PRSC), nonproliferating rheumatoid synovial cells (NPRSC), proliferating nonrheumatoid synovial cells (PNSC), and nonproliferating nonrheumatoid synovial
cells (NP-NSC). The results are presented in Table 1.
Significant differences were observed between proliferating and nonproliferating cultures and between the RSC
and NSC derived cells. In both cell types, the proliferative state was associated with higher levels (P< 0.001, P
< 0.01) of surface plus secreted proteases. This observation has been previously reported with other cell types
(20,27). The NP-RSC contained significantly higher
surface plus secreted protease activity compared t o NPNSC (P< 0.02). Higher levels of surface plus secreted
protease activity were also observed on the P-RSC compared to the P-NSC (P < 0.05). The proliferating plus
nonproliferating rheumatoid synovial cells exhlbited
significantly higher surface plus secreted protease activity compared to the proliferating plus nonproliferating
nonrheumatoid synovial cells (Pc 0.05).
The proliferating cells (RSC plus NSC) also exhibited significantly higher surface plus secreted protease activity compared to the nonproliferating cells
-~
RA 13
Mean f SD
Prolijeraring
RA4
KA5
RA 5
RA 6
RA 1 1
RA 13
*
Mean SD
Total Mean f SD
2.6
1.6
1.2
2.4
1.6 f 0.78
4.1
4.5
5.5
3.8
7.9
3.1
4.8 f 1.71
3.1 f 2,1#
-.
*N
= nonrheumatoid adherent synovial cells; RA = rheumatoid ad-
herent synovial cell.
t One unit of protease activity is defined as the amount of protease activity which solubilizes 1,ooO cpm of TCA soluble 3H-acetyl peptides
after incubation for 180 minutes at 37°C. Each value represents mean
value of protease activity from duplicate plates.
$ Proliferating nonrheumatoid synovial cells significantly higher than
nonproliferating nonrheumatoid synovial cells, P < 0.001.
5 Rheumatoid nonproliferating synovial cells significantly higher protease activity compared to nonproliferating nonrheumatoid synovial
cells (P < 0.02).
7 Proliferating rheumatoid synovial cells significantly higher than
nonproliferating rheumatoid synovial cells, P < 0.01. Proliferating
rheumatoid synovial cells significantly higher than proliferating nonrheumatoid synovial cells, P < 0.05.
tt Rheumatoid synovial cells significantly higher than nonrheumatoid
synovial cells, P < 0.05.
Proliferating rheumatoid synovial cells plus proliferating nonrheumatoid synovial cells exhibit significantly higher surface plus secreted protease activity compared to nonproliferating rheumatoid synovial cells plus nonproliferating nonrheumatoid synovial cells, P <
0.001.
HATCHER ET AL
922
(RSC plus NSC) (P < 0.001). Surface plus secreted protease activity was not related to the passage number of
the proliferating or nonproliferating cells, but was related only to the proliferative state and probably to the
rheumatoid origin of the cells. That is, the cultures of PRSC contained the highest levels of surface plus secreted protease activity.
Secreted protease activity. Secreted protease activity was examined in the RSC and NSC. Synovial
cells were permitted to secrete proteases into serum-free
culture medium for 180 minutes. The medium was removed, centrifuged, and assayed for secreted activity. In
this assay, protease activity associated with the cell surface was not measured. No difference in the secreted
protease activity was observed in proliferating and nonproliferating synovial cells in the limited number of cultures evaluated (Table 2). Secreted protease activity was
observed in only one NSC line. In the rheumatoid cell
lines, higher levels of secreted protease activity (P <
0.01) were observed compared to the 4 NSC lines. In
comparing surface plus secreted protease activity (Table
1) with secreted protease activity alone (Table 2) in proliferating nonrheumatoid and rheumatoid cell lines,
higher levels of protease activity were observed in the
Table 2. Secreted neutral protease activity in rheumatoid and
nonrheumatoid synovial cells
~
Passage
number
Synovial cells*
Nonrheumatoid
Proliferating
Nonproliferating
Proliferating
Nonproliferating
Proliferating
Nonproliferating
Mean f SD
N6
N6
N8
N 12
N 13
N 13
Rheumatoid
Proliferating
Nonproliferating
Proliferating
Nonproliferating
Nonproliferating
Nonproliferating
Nonproliferating
Nonproliferating
Nonproliferating
Mean -+ SD
RA 4
RA 4
RA 5
RA 5
RA 6
RA 10
RA I 1
RA 12
RA 13
~
~
_
Protease activity,
units/105 cellst
0
0
I .8
0
0
0
0.3 k 0.7
2.8
2.3
3.2
I .7
2.9
1.9
I .4
0.8
1.5
2.1 f 0.84~~
* N = nonrheumatoid synovial cells; RA = rheumatoid synovial cells.
t One unit of protease activity is defined as the amount of protease activity which solubilizes 1,OOO cpm of TCA soluble 'H-acetyl peptides
after incubation for 120 minutes at 37°C. Protease activity represents
mean values from duplicate plates.
4- Rheumatoid synovial cells exhibit significantly higher secreted protease activity compared to nonrheumatoid synovial cells, P < 0.01.
Table 3. Extractable acidic protease activity in rheumatoid and
nonrheumatoid synovial cells
~
Cell lines*
Passage
number
~
~~
Protease activity,
units/lO5 cellst
- - - - _ _ _ ~
pH 5.0
pH 6.0
Nonrheumatoid
N3
N5
N6
N8
Mean rt SD
36.5
43.2
15.9
18.5
28.5 f 13.4
26.6
20.4
11.4
14.4
18.2 f 6.7
N3
N5
N6
N8
Mean f SD
Total Mean f SD
20.5
25.0
27.2
26.2
24.7 f 3.0
26.6 f 8.6
7.9
15.9
15.6
14.0
13.4 f 3.7
17.7 f 7.8
Rheumatoid
RA 5
RA 5
RA 6
RA 10
RA 11
RA 13
Mean f SD
53.0
55.4
35.9
46.5
155.0
56.3
67.0 f 43.8
27.1
40.5
30.5
33.3
93.8
60.8
47.7 f 25.6
49.8
66.7
31.1
49.2 f 17.8
62.0 f 34.84-
27. I
47.1
22.4
32.2 13.1
48.5 f 28.68
RA 5
RA 5
RA 6
Mean f. SD
Total Mean f SD
3
4
3
*
~
~~~~
* N = nonrheumatoid synovial cells; RA = rheumatoid synovial cells.
_
_
t One
unit of protease activity is defined as the amount of protease activity which solubilizes 1,OOO cpm of TCA soluble 3H-acetyl peptides
after incubation for 60 minutes at 40°C. Protease activity represents
mean values from duplicate plates.
4- Rheumatoid synovial cells significantly higher than nonrheumatoid
synovial cells, P < 0.05.
§ Rheumatoid synovial cells significantly higher than nonrheumatoid
synovial cells, P < 0.02.
experiments in which a combination of surface associated plus secreted protease activity was measured.
Extractable protease activity. Proteases were
also extracted from proliferating and nonproliferating
RSC and NSC. No significant difference was observed
in the amount of extractable protease activity measured
at pH 5.0, 6.0, 7.0, and 8.0 between proliferating and
nonproliferating cells of both types. Extractable proteolytic enzyme activity measured at pH 5.0 and pH 6.0
is shown in Table 3. No difference was observed in the
proteolytic activity in the extracts at different passages
in the limited number of cells examined. Rheumatoid
cell extracts contained significantly higher protease activity at pH 5.0 (P< 0.05) and at pH 6.0 (P< 0.02). In
comparing protease activity from early passage NSC ex-
NEUTRAL PROTEASE ACTIVITY
tracts at pH 5.0 and pH 6.0, no significant differences
were observed. Extracts from NSC at passage 3,4, and 5
exhibited higher protease activity at pH 5.0 (P< 0.01)
compared to activity at pH 6.0.
Extractable neutral proteolytic activity in rheumatoid and nonrheumatoid synovial cells is shown in
Table 4. At neutral pH, significant differences were observed between the passage number and the amount of
protease activity. In the extracts from NSC, significantly (P< 0.05) higher amounts of neutral protease activity were observed at passage 0 and 1 compared with
extracts from cells at passage 3, 4, and 5.
In the RSC lines, significantly higher amounts (P
< 0.02) of neutral protease activity were also present in
the cell lines at passage 0 and 1 compared with extracts
obtained from cells at passage number 3 and 4. The
early passaged RSC extracts also contained higher levels of neutral protease activity compared with the earlypassaged NSC (P < 0.02).
In comparing the neutral protease activity in the
rheumatoid extracts from all cell lines with the extracts
from all nonrheumatoid cell lines, the extracts from
rheumatoid cell lines had significantly higher levels of
neutral protease activity (P < 0.02). Synovial cells at
passage 0 and 1 (RSC plus NSC) exhibited higher neutral protease activity compared to synovial cells at passage 3, 4, and 5 (RSC plus NSC) (P < 0.01). Nonrheumatoid early passage synovial cells contained
higher protease activity at pH 7.0 (Table 4) compared to
activity at pH 6.0 (P< 0.02) (Table 3) and at pH 8.0 (P
c 0.02) (Table 5). Early passaged RSC had higher neutral protease activity (Table 4) compared to activity at
pH 6.0 (P < 0.05) (Table 3).
Protease activity was compared in extracts from
RSC and NSC at pH 8.0 (Table 5). No significant difference was observed between the passage number and
the protease activity measured at pH 8.0 in RSC and
NSC. The RSC contained elevated cellular protease activity measured at pH 8.0 compared with the extracts
obtained from the NSC (P< 0.02). The RSC at passage
0 and 1 contained higher protease activity at pH 8.0
compared to NSC at passage 0 and 1 (P < 0.05). The
early passaged RSC exhibited significantly more extractable protease activity at pH 7.0 (Table 4) compared
to early passaged RSC measured at pH 8.0 (P< 0.02)
(Table 5).
DISCUSSION
The present investigation demonstrates that cultured adherent RSC possess higher levels of protease
923
Table 4. Extractable neutral protease activity in rheumatoid and
nonrheumatoid synovial cells
-_ -.
Cell lines*
Passage
number
Protease activity,
units/ lo5 cells,
pH 7.0t
Nonrheumatoid
N3
N5
N6
N8
Mean f SD
31.2
37.0
28.1
31.1
3 1.9 & 3.7$
N3
N5
N7
N8
Mean f SD
Total Mean f SD
27.1
14.7
26.0
16.5
21.1 f 6.4
26.5 7.5
Rheumatoid
RA 4
RA 5
RA 5
RA 6
RA 7
RA 8
RA 10
RA 1 1
RA 13
Mean f SD
RA 4
RA 5
RA 5
RA 6
Mean f SD
Total Mean 2 SD
*
53.6
39. I
76.7
77.2
75.4
83.9
44.1
120.3
47.0
68.6 f 25.69
26.0
39.1
24.1
26.0
28.8 f 6.9
56.3 -c 28.61
* N = nonrheumatoid synovial cells; RA = rheumatoid synovial cells.
t One unit of protease activity is defined as the amount of protease activity which soluhilizes 1,OOO cpm of TCA soluble 'H-acetyl peptides
after incubation for 60 minutes at 40°C. Mean values from duplicate
plates.
$Nonrheumatoid synovial cells at passage 0 and 1 contain significantly higher neutral protease compared to nonrheumatoid synovial
cells at passage 3, 4, and 5, P < 0.05.
5 Rheumatoid synovial cells at passage 0 and I contain significantly
higher neutral protease activity compared to nonrheumatoid synovial
cells at passage 0 and 1, P < 0.02. Rheumatoid synovial cells at passage 0 and 1 contain significantly higher neutral protease compared to
rheumatoid synovial cells at passage 3 and 4, P < 0.02.
1Neutral protease activity from rheumatoid synovial cells significantly higher compared to neutral protease from nonrheumatoid synovial cells, P < 0.02.
Neutral protease activity from nonrheumatoid synovial cells plus
rheumatoid synovial cells at passage 0 and 1 significantly higher than
neutral protease activity from nonrheumatoid synovial cells at passage 3 and 4 plus rheumatoid synovial cells at passage 3 , 4 , and 5, P <
0.01.
activity compared to NSC. P-RSC have elevated surface associated plus secreted protease activity compared
to NP-RSC and P-NSC. Several investigators have reported a relationship between cell proliferation and cellular protease activity. Chou et a1 (24) reported that the
HATCHER ET AL
924
Table 5. Protease activity a t pR 8 . 0 i n rheumatoid and
nonrheumatoid synovial cells
Cell lines.
Passage
number
Protease activity,
units/105 cells,
pH 8.0t
Nonrhcumatoid
N3
N5
N6
N8
N9
Mean f SD
15.2
12.6
25.8
28.7
22.5
21.0 f 6.9
N3
N5
N9
N 10
N I1
Mean f SD
Total Mean +- SD
19.1
11.6
26.3
19.7
22.5
20.0 f 5.4
20.5 f 5.8
Rheumatoid
RA 4
RA 5
RA 5
RA 6
RA I
RA 9
RA 10
RA 13
Mean SD
*
RA 4
RA 5
RA 5
RA 6
Mean f SD
Total Mean f SD
0
0
1
0
0
1
I
I
4
3
4
3
21.5
39.1
34.5
69.9
37.1
41.9
36.1
25. I
38.3 f 14.6*
19.7
36.1
28.1
21.1
26.4 f 7.8
34.3 f 13.68
--
N = Nonrheumatoid synovial cells; RA = rheumatoid synovial
Cells.
t One unit of protease activity is defined as the amount of protease activity which solubilizes 1,OOO cpm of TCA soluble 'H-acetyl peptides
after incubation for 60 minutes at 40°C. Mean values from duplicate
plates.
$ Rheumatoid synovial cells at passage 0 and I contain higher protease activity compared to nonrheumatoid synovial cells at passage 0
and 1, P < 0.05.
8 Rheumatoid synovial cells contain higher protease activity compared to nonrheumatoid synovial cells, P < 0.02.
maximal secretion of plasminogen activator occurs
when Swiss 3T3 cells were proliferating. Rohrlick and
Rifkin (25) reported that cultured low passaged embryo
fibroblasts elaborate increasing plasminogen activator
activity before they reach confluence. Bauer (26) observed the maximal secreted collagenase activity shortly
after the skin fibroblasts reached confluence. The secreted collagenase activity decreased in post-confluent
cells.
Surface associated neutral protease activity has
also been observed in a large number of proliferating
cells (20). The maximal surface protease activity was
observed just before, during, or just after mitosis. Nonproliferating cells have decreased surface associated
neutral protease activity (27,28). Cartilage derived factors and heparin which inhibit cell proliferation also inhibit surface associated protease activity (29).
In the present study, it was not possible to differentiate surface associated protease activity from secreted protease activity, since RSC and some NSC secrete protease into the medium under experimental
conditions in which surface protease activity has been
determined in previous studies (20,27). Although RSC
secrete higher amounts of proteases into the medium
than do NSC, no differences in secreted protease activity alone were observed between proliferating and nonproliferating cells (Table 2). significant differences in
protease activity were observed between proliferating
and nonproliferating cells in experiments in which a
combination of surface associated plus secreted protease
activity was measured (Table 1).
T h e results from previous experiments
(20,27,29), together with the results presented on Table
1 and Table 2, suggest that proteases associated with the
cell may be responsible for part of the protease activity
in P-RSC and P-NSC. The functional role of proteases
associated with the membrane is not known. One could
speculate that during proliferation, membrane proteases
are responsible for the degradation of specific proteins
in the extracellular matrix.
During proliferation, specific membrane proteins
could also be degraded by membrane proteases. Linsley
et a1 (30) have reported that epidermal growth factor
(EGF) receptor complex binding undergoes proteolytic
modifications in murine 3T3 cells and human foreskin
fibroblasts. Two distinct proteases, one of unknown
specificity and one similar to trypsin, seem to be involved in the cleavages of EFG-receptor complexes
(31). Recently, Bach et a1 (32) suggested that plasma
membrane proteases control the binding of IgE to specific receptors in rat peritoneal mast cells. It is not understood why P-RSC possess higher surface plus secreted protease activity than do P-NSC. Adherent RSC
dispersed by proteolysis of rheumatoid synovium consist of at least two cell types (9-12). The majority of the
population are large stellate cells with many dendritic
processes lacking macrophage markers. Small adherent
monocyte macrophages with receptors for the Fc portion of the immunoglobulin constitute the second type
of adherent cell in primary cultures. Serial passaged synovial cells have less than 2% of the small adherent
cells. Primary cultures of NSC have fewer small adher-
925
NEUTRAL PROTEASE ACTIVITY
ent cells compared to primary adherent RSC (unpublished results). No significant difference was observed in
surface plus secreted protease activity between the primary cultures containing both types of cells and the serial passaged cells consisting of mainly large stellate
cells. The enhanced levels of surface plus secreted protease activity in the proliferating rheumatoid and nonrheumatoid cells were not related to the presence of the
small adherent cells. It was not possible to determine if
different populations of stellate cells exist between the
rheumatoid and nonrheumatoid cells.
Proteolytic enzymes were extracted from proliferating and nonproliferating synovial cells. No significant difference was observed between the extracted protease activity in proliferating or nonproliferating
adherent synovial cells. Rheumatoid synovial cell extracts contained elevated protease activity measured at
pH 5.0, 6.0, 7.0, and 8.0. Lysosomal enzymes have been
proposed in the destruction of cartilage matrix (33,34).
The protease activity measured at pH 5.0 and 6.0 are
probably due to lysosomal enzymes (33) since lysosomal
enzymes are increased in rheumatoid synovia (34).
Neutral proteases have also been implicated in
the degradative process of articular cartilage (16- 18).
Werb et a1 (35) isolated a neutral metalloprotein protease from rabbit synovial fibroblasts which degrade bovine nasal proteoglycan and azo-casein. In the present
study, human synovial adherent cells contain large
amounts of extractable neutral protease when assayed
with 'H-acetyl casein. The amount of neutral protease
was related to the passage number of the cells. Nonrheumatoid cells at passage 0 and 1 contain higher neutral protease activity compared to nonrheumatoid cells
at passage 3, 4, and 5. Rheumatoid early passaged cells
also exhibited enhanced neutral protease activity. The
differences in extractable protease activity in the early
passaged cells compared to serially passaged cells are
probably due to the presence of different types of cells.
Primary cells contain both large stellate cells and small
monocyte macrophages, while the serially passaged cells
contain mainly large stellate cells (9-12). In nonrheumatoid cells, difference in extractable protease activity between pH 5.0 and 6.0 was only observed in serially passaged cells. Enhanced protease activity at pH 7.0
compared to pH 6.0 and 8.0 was observed in primary
and first passaged cells. The differences in protease activity measured at neutral pH were not seen in synovial
cells at passage 3, 4, and 5. The results suggest that the
monocyte-macrophages contribute either directly or indirectly to the enhanced neutral protease levels in extracts of rheumatoid and nonrheumatoid cells.
ACKNOWLEDGMENTS
The authors are indebted to Drs. Arthur Grayzel, Peter Barland, and David Hamerman for constructive criticism
of this manuscript and helpful discussion. The excellent technical contribution of Rita Craig is greatly appreciated. The
secretarial assistance of Leslie Leibowitz is gratefully acknowledged.
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