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Septic arthritis.

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533
SEPTIC ARTHRITIS
Staphylococcal Induction of Chondrocyte Proteolytic Activity
RILEY J. WILLIAMS, 111, R. LANE SMITH, and DAVID J. SCHURMAN
We report herein that cartilage proteolytic activity increased in bovine and rabbit articular cartilage
after treatment with a purified staphylococcal culture
medium or intraarticular infection with Staphylococcus
aureus. Staphylococcal culture medium increased the
release of gelatinolytic, collagenolytic, and caseinolytic
activity into the medium of isolated chondrocytes or
cartilage organ culture. The proteolytic activities were
determined in assays using radiolabeled substrate and
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staphylococcal culture medium was proteolytically inactive by both assay techniques. RNA synthesis
of isolated chondrocytes was unaffected by staphylococcal culture medium, whereas overall protein synthesis
was inhibited by 84%. An analysis of extracts of Staphylococcus aureus-infected rabbit knee cartilage by substrate gels showed increased gelatinolytic and caseinolytic activity compared with extracts of uninfected knee
cartilage. Our data suggest that the rapid loss of proteoglycan and persistent degradation of cartilage in
staphylococcal septic arthritis is due to the production
and activation of chondrocyte proteases.
Septic arthritis continues to be a serious problem in urban medical centers ( I ,2) and is recognized as
From the Orthopedic Research Laboratory, Stanford University School of Medicine, Stanford, California.
Supported in part by NIH grants AR-30796 and AR-26833.
Mr.Williams’ work was supported in part by the Stanford Medical
Student Scholars Program.
Riley J. Williams, 111, BS; R. Lane Smith, PhD; David J .
Schurman, MD.
Address reprint requests to R. Lane Smith, PhD, 300
Pasteur Drive, R171, Stanford, CA 94305-5326.
Submitted for publication July 13, 1989; accepted in revised
form November 29, 1989.
Arthritis and Rheumatism, Vol. 33, NO. 4 (April 1990)
a complicating factor in rheumatoid arthritis and other
chronic arthritic conditions (3). Although most joint
infections are acquired hematogenously, direct infection can result from deep wounds, surgical procedures, and intraarticular joint injections ( 2 ) . In nongonococcal septic arthritis, Staphylococcus aureus is
the organism most frequently found by culture (64%);
in 25% of the patients positive for S aureus, the
bacteria are methicillin resistant (1). Sterile “postinfectious” arthritis provides the potential for residual
joint damage (43).
In an animal model of staphylococcal infectious
arthritis, cartilage degradation was shown to continue,
despite early antibiotic treatment (5). Infection of
rabbit knees with S aureus was shown to cause a rapid
with
),
a loss of
loss of cartilage proteoglycan (PG) (a
as much as 40% of the total PG from the cartilage
matrix within the first 48 hours after infection (8). Loss
of collagen becomes significant between the second
and third weeks after infection (8). Antibiotic treatment begun within 24 hours of infection decreases
collagen loss but fails to prevent loss of PG from the
matrix (5).
In vitro, S aureus or S aureus-conditioned
culture medium induces cartilage degradation in viable
cartilage explants (9). Cartilage degradation is marked
by a loss of partially degraded PG, which is consistent
with increased proteolytic activity. PG release from
cartilage by S aureus or staphylococcal culture medium is significantly reduced by freeze-thawing cartilage (9). Killed bacteria have no effect on cartilage
degradation. Staphylococcus-induced PG release is
dependent on chondrocyte metabolism and can be
blocked by inhibitors of RNA and protein synthesis,
i.e., actinomycin D and cycloheximide, respectively
WILLIAMS ET A L
534
(10). These data suggest that Staphylococcus-induced
articular cartilage degradation is dependent upon activation of gene expression and subsequent synthesis of
a chondrocyte-derived protease($ or protease activator(s).
In the experiments reported herein, we quantitated the effects of S aur eus and purified staphylococcal culture medium (SCM) on RNA and protein synthesis and on collagenase, caseinase, and gelatinase
activity. We used isolated bovine articular chondrocytes or bovine cartilage slices t o analyze SCMinduced protease activity. Protease activity induced
by S aureus was analyzed using cartilage removed
from Staphylococcus-infected rabbit knees.
I
MATERIALS AND METHODS
Purification of staphylococcal culture medium. SCM
was purified from the culture medium of a 4-day growth of S
aureus (10). Bacteria were grown in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco, Grand Island, NY) without
phenol red. SCM was sterilized by ultrafiltration (0.22 pm),
concentrated on a hollow fiber membrane filter (>IO,OOO
MW pore), butanol extracted, and desalted by G-25 gel
filtration chromatography. The range of protein concentration in SCM was 50-70 nglml.
Preparation of cartilage cells. Adult bovine articular
chondrocytes were prepared from cartilage slices dissected
from the radiocarpal joints of 2-3-year-old animals, as previously described (1 1). Chondrocytes were dissociated by
treatment with type I1 and type IV collagenase (Worthington, Freehold, NJ), at a concentration of 0.6 mg/ml each, in
DMEM. Following overnight incubation at 37°C in 7.5% CO,
and 100% humidity, dissociated cells were diluted 2.7-fold
with Dulbecco’s phosphate buffered saline (PBS) without
Mg++ or C a ++ (Gibco) and collected by centrifugation at
450g for 20 minutes. Cells were washed in PBS without
Mg++ or C a ++ (40 ml) and in DMEM (40 ml), resuspended
in 10 ml of DMEM, filtered through Nitex (TETKO, Monterey Park, CA), and counted. Viability was greater than
95% by trypan blue exclusion.
Chondrocytes were plated at high density (1 x lo5/
cm2) in 2 ml of a 1:1 solution of DMEM and Ham’s F-12
medium containing 3% fetal bovine serum. Cells were cultured in 10 x 35-mm dishes as described above. After 2 days
in culture, the 3% serum medium was removed, the cells
were washed 3 times with 2 ml of PBS each time, and were
then transferred to serum-free medium. Serum-free medium
consisted of a 1:l mixture of DMEM and Ham’s F-12
medium:gentamicin (50 CLg/rnl) (12). After 2 days in serumfree culture, plated cells were challenged with 200 pl of SCM
and incubated as described. Control cells were not challenged.
Organ culture of cartilage slices. Full-thickness slices
of bovine articular cartilage (I0 mg wet weight) were obtained as described above and were distributed in sterile
24-cluster microwell culture plates (Costar, Cambridge,
MA). Each slice was challenged with 50 pl of SCM and
maintained in 500 pl of DMEM without serum, as with the
cell cultures (9).
Induction of staphylococcal infectious arthritis. Arthritis was induced in the right knees of female New Zealand
white rabbits inoculated with 9.2 x lo4 colony-forming units
of S aureus (5). Antibiotic treatment was started 24 hours
after inoculation and was administered for 10 days (5). The
animals were killed at 3 weeks postinfection, and the articular cartilage on all joint surfaces was removed for biochemical analysis. Cartilage from uninfected left knees served as
the control.
Biosynthetic labeling of cellular RNA and cellular
protein. Total RNA synthesis was quantitated over a 24-hour
period using 10 pCi/ml of ’H-uridine (specific activity 28.5
Ci/rnmole; New England Nuclear, Boston, MA), which was
added simultaneously with the SCM. After 24 hours, the
medium was removed and retained: the cells were then
harvested in 2 ml of PBS and sonically disrupted. Cellassociated protein synthesis and secreted protein synthesis
were quantitated using 10 pCi/ml of ,H-serine (specific
activity 14.4 Ci/mmole; New England Nuclear), which was
added to cell culture plates 48 hours following the challenge
with SCM.
After a second 48-hour period, the culture medium
was collected; the cells were harvested in 2 ml of PBS and
sonically treated. Aliquots of sonically treated cells were
precipitated with trichloroacetic acid (TCA; 10% volume/
volume) to isolate radiolabeled RNA or radiolabeled cellassociated protein. Culture medium was precipitated with
TCA to isolate radiolabeled secreted protein. Precipitates
were collected on Millipore filters (HAWP 0.45 pm; Millipore, Bedford, MA). Filters were placed in 6 ml of OptiFluor (Packard Instrument Company, Downer’s Grove, IL),
and radioactivity was measured by scintillation counting
using a Beckman LS-7500 scintillation counter (Beckman
Instruments, Irvine, CA).
Autoradiographic gel analysis. 3H-serine-labeled proteins in medium samples were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE;
12% gels) (13). Aliquots of medium were treated with TCA,
and the precipitates were collected by centrifugation at
39,OOOg for 10 minutes. Pellets were treated with acetone,
suspended in SDS sample buffer, and boiled. Sixty microliters of the resuspension was loaded on SDS-polyacrylamide
gels (12%). After electrophoresis, gels were immersed in 100
ml of 100% DMSO (Sigma, St. Louis, MO). The DMSO
solution was changed 3 times at 20-minute intervals. The gel
was subsequently treated with 4 volumes of a mixture of
22.5% Omnifluor (New England Nuclear) in 100% DMSO
for 3 hours. Autoradiographs were prepared from dried
gels with preflashed X-Omat AR film (Kodak, Rochester,
NY) (13).
Assay of casein-degrading activity. Radiolabeled I4C@casein (15.7 pCi/mg) was obtained from Sigma. The assay
procedure for casein-degrading activity was similar to that
described by Golds et al (14). Cell medium from SCMtreated cells and control cells was activated with 1 mM
APMA for 1 hour at 37°C. Reaction mixtures contained 20 p1
of ‘‘C-labeled casein (2.6 x lo5 counts per minute), 50 pl of
assay buffer (100 rnM Tris HCI, pH 7.4,0.4M NaCI, 20 mM
CaCI,, and 0.04% NaN,), and 150-pI samples of chondrocyte
culture medium (total volume 220 PI).
STAPHYLOCOCCUS AND SEPTIC ARTHRITIS
Reactions were performed for 18 hours at 37°C and
then terminated by adding 100 p-1 of unlabeled casein (3
mg/ml) and 150 pl of 10% TCA (volumelvolume). The
mixtures were centrifuged at 4°C for 15 minutes at 7,000g.
Supernatant (200 pl)was collected from each mixture, added
to 6 ml of Opti-Fluor, and radioactive counts were determined using scintillation counting. Enzyme activity was
determined by the release of TCA-soluble radioactivity.
Maximal substrate degradation was estimated using 100 ng
of trypsin (15).
Assay of collagen-degradingactivity. Radiolabeled rat
type I collagen (1 .O mCi 'H per mg) was obtained from New
England Nuclear. The assay procedure for radiolabeled
collagen degradation was similar to that described by Galloway et al (16). Cell medium from SCM-treated cells and
control cells was activated with APMA ( 1 mM) for 1 hour at
37°C. Reaction mixtures contained 1 pl of 3H-labeled rat
type I collagen, 50 pI of assay buffer (50 mM Tris HCI, pH
7.5, and 10 mM CaCI,), 149 pl of H,O, and SO-pI aliquots of
SCM-treated cell medium, giving a final volume of 250 pl.
Reactions were carried out at 37°C for 18 hours. At the end
of the assay interval, undigested collagen was removed by
centrifugation at 10,OOOg for 15 minutes. One hundred fiftymicroliter aliquots of the supernatant were placed in 6 rnl of
Opti-Fluor, and radioactivity was measured. Maximal substrate degradation was estimated using 100 ng of type I1
bacterial collagenase (Worthington).
SDS substrate gel analysis. To determine the molecular weights of bands with enzymatic activity, aliquots of
medium from isolated chondrocytes and organ culture were
electrophoresed on SDS substrate gels containing 1 mg/ml of
gelatin or casein, as described by Chin et al (17). Chondrocyte medium was tested with and without overnight incubation with 0.5 mM APMA to activate latent protease (18).
Aliquots of medium were diluted with 4 x sample buffer (10%
SDS, 4% sucrose, 0.25M Tris HCI, pH 6.8, and 0.1%
bromphenol blue), but were not boiled, and were separated
on a 10% SDS substrate gel at 4"C, with a 20-mA constant
current. After electrophoresis, SDS was removed by shaking
the gel in 2.5% Triton X-100 for 30 minutes at 25°C. To
facilitate the digestion of gelatin and casein by proteases, the
gels were incubated for 18 hours at 37°C in 50 mM Tris HCI
buffer, pH 8, containing 10 mM CaCI, and 0.02% NaN,.
After incubation, the gels were stained with 0.5% Coomassie
535
Figure 1. Effect of staphylococcal culture medium (SCM) on secretion of bovine articular chondrocyte proteins. Chondrocytes were
isolated and grown to confluence in medium containing 3% serum.
After repeated washes with phosphate buffered saline, the medium
was removed and replaced with serum-free medium. After 2 days in
serum-free culture, chondrocytes were treated with SCM. Two days
following the challenge with SCM, cells were labeled for 48 hours
with 'H-serine, and secreted proteins were analyzed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lanes 1 and 2 represent cultures not challenged with SCM;
lanes 3 and 4 represent cultures challenged with SCM. Each lane
was loaded with a 30-pI sample of medium. Molecular weight
markers are shown at the left.
Table 1. Quantitation of medium and cell-associated protein synthesis by incorporation of 'H-serine*
No. of
plates
analyzed
Culture medium
Untreated
SCM treated
Cell-associated
Untreated
SCM treated
Mean t SEM
cpm ( x lo-'/
culture)
96
inhibition
12
12
784 t 30
122 2 8
85
6
6
335 2 9
58 2 3
83
-
* Each culture plate contained approximately lo6 cells. The percentage of inhibition of protein synthesis by staphylococcal culture
medium (SCM)-treated cells versus untreated cells was significant
for both conditions (P< O.OOO1 by Student's 2-sample (-test).
blue R250 in 30% isopropyl alcohol with 10% acetic acid and
destained in water. The SCM preparation was also analyzed
for proteolytic activity as described above.
I n vivo protease production by cartilage from
Staphylococcus-infected rabbit knees was analyzed using
gelatin and casein substrate gels. Cartilage from both the
right and left knee joints of rabbits was dissected and placed
in 500 pI of 4x sample buffer overnight. Dry weights for
uninfected control cartilage ranged from 3.76 x
gm to
6.68 x lop2 gm; Staphylococcus-infected cartilage dry
weights ranged from 1.20 x lop2 gm to 2.25 x lop2 gm.
Proteases from the cartilage extracts were separated by SDS
substrate gel analysis without further modification.
536
WILLIAMS ET AL
Table 2. Collagen- and casein-degrading activity of medium from
isolated chondrocytes*
No. of plates
analyzed
Collagenolytic activity
Untreated cell
medium
SCM-treated cell
medium
Caseinolytic activity
Untreated cell
medium
SCM-treated cell
medium
Mean ? SEM cpm
( X IO-’/cuIture)
8
413
?
144
12
2,037
5
l5Ot
9
24.6
?
0.9
14
(Figure 2) and caseinolytic (Figure 3) activity were
noted in the 50-60-kd range. These bands were absent
or rarely found in medium from control cells. APMA
treatment of medium samples enhanced the proteolytic activity resulting from SCM treatment (Figures 2
and 3). APMA treatment also caused 2 of the 3
50-55-kd proteinase bands to shift electrophoretic
51.8 -t l.4$
* Each culture plate contained approximately lo6 cells. The collagenolytic activity was the radioactivity remaining in the supernatant
after centrifugation; the caseinolytic activity was the radioactivity
remaining in the trichloracetic acid-soluble supernatant. Degradation with 100 pl of bacterial collagenase (100 ng/pl) was 6.5 x 104
cpm; degradation with 100 p1 of trypsin (100 ng/pI) was 3.6 x lo4
cpm. SCM = staphylococcal culture medium.
t P < 0.01 by Student’s 2-tailed 2-sample z-test.
t P < O.OOO1 by Student’s 2-tailed 2-sample t-test.
RESULTS
Quantitation of cellular RNA and protein synthesis. Analysis of total RNA synthesis, according to
’H-uridine incorporation, showed no statistical difference between cells treated with SCM and untreated
cells. Analysis of protein synthesis, using ’H-serine
incorporation, however, showed an 85% decrease in
the synthesis of proteins released into the culture
medium by cells treated with SCM versus untreated
cells (Table 1). Cell-associated protein synthesis was
inhibited by 83% in SCM-treated cells versus untreated cells (Table 1). Overall protein synthesis was
inhibited by 84%. The proportion of newly synthesized
proteins released into the medium was 70% for untreated control cells and 68% for SCM-treated cells.
Autoradiography. ’H-serine-labeled proteins
were separated by SDS-PAGE. Radiolabeled proteins
from SCM-treated cells and control cell medium
yielded distinct protein banding patterns (Figure 1).
The most notable difference between the 2 profiles was
the appearance of 2 protein bands in the 50-55-kd
range and a single band in the 34-36-kd range in
SCM-treated cell medium.
Caseinase and collagenase assays’
casein and collagen degradation assays dUTlonstrated
that medium from isolated cartilage cells treated with
SCM possessed twice as much caseinase activity and 5
times as much
activity as did the medium
from untreated cells (Table 2).
Substrate gels. In medium from SCM-treated
cells, prominent proteinase bands with gelatinolytic
Figure 2. Sodium dodecyl sulfate (SDS) substrate gel analysis of
the gelatin-degrading activity of medium from isolated bovine chondrocytes challenged with staphylococcal culture medium (SCM).
Samples from the same pools used in Figure 1 were electrophoresed
on an SDS substrate gel containing I mg/ml gelatin and processed as
described in Materials and Methods. Lysis zones indicate gelatinolytic activity. Lane 1 (untreated) and lane 2 (APMA activated)
represent untreated chondrocyte medium; lane 3 (untreated) and
lane 4 (APMA activated) represent SCM-stimulated chondrocyte
medium. Molecular weight markers are shown at the right.
STAPHYLOCOCCUS AND SEPTIC ARTHRITIS
Figure 3. SDS substrate gel analysis of the casein-degrading activity of medium from isolated bovine chondrocytes challenged with
SCM. Samples from the same pools used in Figure 1 were electrophoresed on an SDS substrate gel containing I mglml casein and
processed as described in Materials and Methods. Lysis zones
indicate caseinolytic activity. Lane 1 (untreated) and lane 2 (APMA
activated) represent untreated chondrocyte medium; lane 3 (untreated) and lane 4 (APMA activated) represent SCM-stimulated chondrocyte medium. Molecular weight markers are shown at the right.
See Figure 2 for definitions of abbreviations.
mobility to the 4148-kd range. Medium samples from
both control and SCM-treated cells that were separated on gelatin substrate gels showed a zone of
degradation in the area of 92 kd. With SCM treatment,
537
Figure 4. SDS substrate gel analysis of the gelatindegrading activity
of medium from bovine cartilage explants in serum-free organ culture
challenged with SCM. Bovine articular cartilage was dissected from
radiocarpal joints; slices were placed in microwell plates and maintained in Dulbecco’s modified Eagle’s medium without serum. Cartilage explants were challenged with SCM and maintained for 4 days.
Control cartilage was not challenged. Medium from these cultures was
electrophoresed on SDS substrate gels containing 1 mg/ml gelatin and
processed as described in Materials and Methods. Lysis zones indicate
gelatinolytic activity. Lane 1 (untreated) and lane 2 (APMA activated)
represent medium from untreated cartilage rudiments; lane 3 (untreated) and lane 4 (APMA activated) represent medium from SCMstimulated cartilage rudiments. SDS substrate gel analysis of this
medium using casein substrate yielded results similar to those observed
in Figure 3. Molecular weight markers are shown at the right. See
Figure 2 for definitions of abbreviations.
538
Figure 5. Sodium dodecyl sulfate ( S I X ) substrate gel analysis of
the gelatin-degrading activity of isolated rabbit knee cartilage extracts after induction of staphylococcal infectious arthritis. Articular
cartilage from control and infected knees was extracted overnight in
4 x sample buffer as described in Materials and Methods. The
extracts were electrophorescd on SDS gels containing 1 mg/nil
gelatin. Lanes 1 and 2 represent control cartilage extracts: lanes 3
and 4 represent Srrrphylococctcs-infected cartilage extracts. Fourfold more control cartilage extract was loaded on substrate gels than
on Sraphylococcus-infected cartilage extracts. The zones of lysis
indicate gelatinolytic activity. Molecular weight markers and arrows
demonstrating those Lones of activity. which were prominent in the
extractc of Srup/zy/ococcic.r-infccted cartilage. are shown at the
right.
the gelatinolytic zone appeared more extensive. AIthough other proteinase bands appeared on these gels,
which were derived from control and experimental
medium, they were not dependent upon SCM stimulation. The SCM showed no proteolytic activity when
subjected to substrate gel analysis.
Substrate gel analysis of medium from SCMtreated cartilage organ culture slices in serum-free
medium showed banding patterns similar to those
WILLIAMS ET AL
obtained for cells in monolayer (Figure 4).The zone of
gelatin degradation noted at approximately 92 kd with
medium from control cells in monolayer was less
notable in medium obtained from control organ culture
slices. However, the medium from cartilage slices
challenged with SCM exhibited gelatinolytic degradation at approximately 92 kd. Upon treatment with
APMA, this gelatinolytic zone shifted to the 78-80-kd
range (Figure 4).
Substrate gel analysis of articular cartilage extracts from S aureus-infected rabbit knees demonstrated unique banding patterns when compared with
extracts of uninfected knee cartilage. Extracts of infected knee cartilage exhibited numerous bands with
gelatinase activity that were not found in extracts of
uninfected knees (Figure 5). Staphylococcus-infected
cartilage was characterized by the appearance of 2
bands in the 25-30-kd range and the presence of
prominent proteolytic bands in the ranges of 50-55 kd,
65-70 kd, and 9&92 kd. Although 4 times more control
cartilage extract than Sraphylococcus-infected cartilage extract was loaded on the substrate gels, the
amount of proteolytic activity noted in Sruphylococcusinfected extracts was qualitatively greater.
Casein substrate gel analysis of Sfuphylococcusinfected rabbit cartilage extracts also resulted in the
appearance of unique proteinase banding (Figure 6).
Proteolytic bands, which were not present in cartilage
from uninfected knees, appeared in the ranges of 15-17
kd, 25-30 kd, 4 0 4 2 kd, and 50-55 kd, The low molecular
weight proteases in the ranges of 1S-17kd and 2S30 kd
are proteolytic bands that were not observed after
SCM stimulation of cultured bovine chondrocytcs or
cartilage slices.
DISCUSSION
In this study, we showed that there is a major
alteration in proteolytic activity in bovine and rabbit
articular cartilage both after treatment with purified
staphylococcal culture medium and after intraarticular
infection with Sruphylococcus aureus. SCM treatment
of bovine chondrocytes inhibited overall protein synthesis by 84%. and SCM increased the release of
proteases with collagenolytic, gelatinolytic, and caseinolytic activity by bovine chondrocytes in monolayer
and viable organ cultures. The proteases released into
the culture medium were either latent or inhibited, as
demonstrated by APMA activation. Staphylococcal
infection of rabbit articular cartilage in vivo resulted in
a significantly increased expression of proteolytic enzymes as well as the production of proteases, which
STAPHYLOCOCCUS AND SEPTIC ARTHRITIS
were not observed in uninfected cartilage. These proteases persisted in the cartilage for at least 11 days
after eradication of S aureus with antibiotics.
When articular cartilage undergoes proteolytic
degradation, 2 major constituents of the cartilage
matrix serve as substrates: collagen and proteoglycan.
A number of investigators have documented the appearance of collagenase and stromelysin, a metalloproteinase, after interleukin- 1 treatment of rabbit
chondrocytes (19,20) and phorbol ester treatment of
rabbit synovial fibroblasts (17). Collagenase and
stromelysin are the major neutral proteinases produced by fibroblasts (21-24); both of these enzymes
are secreted simultaneously upon induction (17).
Stromelysin has been implicated in the degradation of
proteoglycans and other noncollagenous proteins of
the extracellular matrix (16). Recent studies have
shown that the latent and active forms of the neutral
proteoglycan-degrading metalloproteinase of human
articular cartilage match those of stromelysin (matrix
metalloproteinase-3) (25). Neutral metalloproteinases
capable of degrading proteoglycans, gelatin, and
casein are present in medium conditioned with human
fibroblasts (26), rabbit fibroblasts (2 1-23), human rheumatoid synovium (27), chondrocytes (28-31), and cartilage extracts (32).
The proteolytic activities observed in the
present studies, after Staphylococcus infection, may
be present in normal joints, but inhibited. Other investigators have used 12-O-tetradecanoylphorbol13acetate (TPAtstimulated rabbit brain capillary endothelial cells to demonstrate that induced metalloproteinases
are regulated by endogenous inhibitors (33). These
studies suggest that coordinated synthesis of proteolytic enzymes and their specific inhibitors may represent a mechanism for protease regulation in vivo (33).
Our studies show the consistent appearance of
a large band of gelatin degradation at approximately 92
kd, analogous to the gelatinase activity reported by
Herron et al (33) for TPA-stimulated rabbit brain
capillary endothelial cells. Gelatinase degrades native
type V collagen and is a latent, neutral metalloproteinase (34-36). Rabbit bones (37), fibroblasts (21), and
macrophages (34) secrete gelatinases into culture medium. Both the SCM challenge in vitro and S aureus
infection in vivo induced gelatinolytic activity. In
organ culture, gelatinolytic activity was increased with
SCM treatment of cartilage, whereas gelatinase activity in untreated cartilage was less apparent. However,
both the SCM-treated and the untreated isolated chondrocytes showed the presence of the 92-kd gelatinolytic band on substrate gels. Sapolsky et a1 (38)
539
Figure 6. Sodium dodecyl sulfate (SDS) substrate gel analysis of
the casein-degrading activity of isolated rabbit knee cartilage extracts after induction of staphylococcal infectious arthritis. Samples
from the same pool used in Figure 5 were electrophoresed on an
SDS substrate gel containing 1 mg/ml casein and processed as
described in Materials and Methods. Lane 1 represents uninfected
cartilage extracts: lanes 2 and 3 represent Sraphylococcus-infected
cartilage extracts. Molecular weight markers and arrows indicating
zones of caseinolytic activity, which were prominent in the extracts
of Sruphylococcus-infected cartilage, are shown at the right.
showed that articular chondrocytes in monolayer culture release latent neutral “gelatinase-like” activity,
which after activation with APMA, degrades type I
collagen and tropocollagen fragments of human type I1
collagen. Initial plating of the cells in 3% serum may
result in a serum component, such as plasminogen
activator (39), causing an increase in background
gelatinase activity in control and experimental chondrocytes, independent of SCM treatment (40).
Substrate gel analysis of extracts of cartilage
obtained from S aureus-infected knees demonstrated
that protease induction occurs in vivo. The substrate
540
WILLIAMS ET AL
degradation observed in Figures 5 and 6 showed that
the proteolytic activities in cartilage were elevated
after infection. Control cartilage extracts showed little
proteolytic activity. Thus, the production and activation of proteases with gelatinolytic and caseinolytic
activity rapidly follows joint infection with Staphylococcus. Moreover, increased protease activity persists, despite early antibiotic treatment. Synovial or
leukocytic proteases may also contribute to the increased levels of proteolytic activity observed in Staphylococcus-infected cartilage extracts.
SDS substrate gel analyses of untreated control
chondrocyte medium or uninfected control cartilage
extracts also demonstrated the presence of some baseline proteolytic bands similar to those noted in SCM or
S aureus-treated samples. This result is expected,
because gel electrophoresis separates existing proteases from their endogenous inhibitors. In addition,
latent enzymes were activated by APMA treatment,
which shifted the electrophoretic mobilities of proteolytic activities. However, in some instances, such as
that shown in Figure 2, overnight treatment with
APMA appeared to diminish gelatinase activity as
well.
In pathologic conditions such as infectious arthritis, it is possible that S aureus increases protease
activity by inactivating inhibitors or by producing an
activator of latent proteases. The rapid loss of proteoglycan and persistent destruction of cartilage that
occurs in staphylococcus infectious arthritis (5-8)
could be explained by the production and activation of
proteases noted in our experiments.
This study demonstrated that a purified staphylococcal culture medium stimulated increased proteinase secretion by cartilage cells in vitro, despite an 84%
inhibition of total protein synthesis. Comparable increases in protease activity were observed in an in
vivo model of staphylococcal infectious arthritis. The
SCM-induced secreted proteinases were chondrocyte
derived and exhibited significant collagenolytic, gelatinolytic, and caseinolytic activity. These proteases, in
their active forms, may contribute to the rapid onset of
cartilage matrix degradation noted in staphylococcal
infectious arthritis.
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