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Metabolic changes in rabbit articular cartilage due to inflammation.

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199
METABOLIC CHANGES IN RABBIT
ARTICULAR CARTILAGE DUE TO
INFLAMMATION
KALINDI DESHMUKH and SUSAN HEMRICK
The effects of inflammation on the articular cartilage of rabbit knee joints were studied. The inflammation
was induced by intrasrticular injections of croton oil or
rabbit peritoneal leukocyte lysates. An increase in the
activities of various lyspsomal enzymes was observed in
the synovial fluid as well as in the cartilage of the inflamed
joints. Loss of proteoglycans, increased rate of degradation of collagen and proteoglycans, and increased
rate of their synthesis were evident in the treated cartilage.
The rate of uptake of SH-thymidinewas also increased. A
significant change was observed in the type of collagen
synthesized by these explants in vitro. In addition to
synthesizing their characteristic Type I1 collagen, the
cartilage explants from the treated joints synthesized
Type I collagen.
Involvement of articular cartilage in degenerative
diseases of joints is well known. Besides the systemic or
the other unknown factors that may be responsible for
triggering the degenerative process, two principal mechanisms have been postulated for the joint destruction in
osteoarthritis or rheumatoid arthritis. The mechanical
From the Department of Physiology, Lilly Research Laboratories, Indianapolis, Indiana 46206.
Kalindi Deshmukh, Ph.D.: Senior Biochemist, Lilly Research
Laboratories; Susan Hemrick, M.S.:Associate Biochemist, Lilly Research Laboratories.
Address reprint requests to Dr. Deshmukh.
Submitted for publication July 24, 1975; accepted August 7,
1975.
Arthritis an4 Rheumatism, V d . 19, No. 2 (March-April 1976)
breakdown due to stress in the weight-bearing areas of
the joint together with the enzymatic degradation of the
matrix components results in erosion of the cartilage
and deformities of the joint.
Early changes in the osteoarthritic cartilage are
associated with a diffuse increase in the number of cells
and loss of proteoglycans with no significant change in
the collagen content (1-3). As the disease progresses
there is a marked decrease in chondroitin sulfate content and the length of polysaccharide chains (4). Metabolic studies indicate an active degradation of cartilage
matrix with a concomitant increase in the uptake of SHglycine and S6SQ, (1). The increase in the rate of synthesis af proteoglycans and cell replication is an indication of the attempt of the repair process.
The collagen content of osteoarthritic cartilage,
as determined by hydroxyproline estimations, does not
differ significantly from that of normal cartilage. Recent
studies by the authors, however, suggest that the
variation occurs in the type of collagen synthesized ( 5 ) .
Whereas normal articular cartilage explants synthesize
only Type I1 collagen, comprised of 3a1(II) chains, osteoarthritic cartilage also synthesizes a more ubiquitous
form, Type I collagen, containing 2al(I) chains and the
laa chain typical of skin and fibrous tissue.
The mechanism underlying the changes in the
metabolic pattern is not fully understood. Increased
levels of lysosomal enzymes encountered in the degenerative conditions (6-10) may play a significant
role in degradation of cartilage matrix. Such enzymic
DESHMUKH AND HEMRICK
200
degradation of cartilage, together with t h e different type
of collagen synthesized, could lead to t h e structural a n d
functional defects o f t h e tissue.
Several a t t e m p t s have been made t o c r e a t e an
experimental model t h a t would simulate t h e h u m a n degenerative diseases of j o i n t s in pathology a n d clinical
observations. Such models include immobilization,
compression, or induction of surgical defects (1 I), a n d
those involving intraarticular injections of materials
such as papain (12), filipin, or streptolysin S (1 3,14).
In the present c o m m u n i c a t i o n t h e a u t h o r s describe the metabolic changes due t o inflammation in t h e
articular cartilage of rabbit k n e e joints. The inflammation was induced and maintained by intraarticular
injections of dilute aqueous suspensions of croton oil
or rabbit peritoneal leukocytes.
MATERIALS AND METHODS
Induction of Inflammation in Rabbit Joints. Male
New Zealand white rabbits weighing 1-2 kg were used for the
studies. These rabbits were young and not fully matured.
Inflammation was induced in the knee joints by intraarticular
injections of a ) 0.25 ml of a 0.5% suspension of croton oil
mixed with 2500 units of penicillin and 2.5 mg of streptomycin
(Gibco), or b) 0.25 ml of rabbit peritoneal leukocyte lysate
(containing approximately 4 X 106 cells/mI) mixed with penicillin and streptomycin.
The peritoneal leukocyte preparation was made by
injecting 20 ml of 1% glycogen into the rabbit peritoneum and
collecting the exudate after 24 hours in magnesium-free KrebsRinger phosphate buffer, pH 7.4, containing 0.1% bovine serum albumin (15). The exudate was passed through a silk
screen and centrifuged at 600 X g for 5 minutes; the pellet was
suspended in the same buffer and centrifuged. The cells were
resuspended in the buffer and counted using a Coulter counter.
The cell lysates were made by repeated freezing and thawing.
The suspension of croton oil and the leukocyte lysate
were filtered through a sterile Swinnex filter before use. The
penicillin-streptomycin solution was added to minimize further the chances of introducing an infection in the joint. It
should be added at this point that the control rabbit joints
injected with penicillin-streptomycin solution alone did not
show any changes similar to the experimental joints.
The injections of croton oil o r leukocytes were repeated on the fifth day after the first injection and the animals
were sacrificed after 5-7 more days. Both agents were able to
produce changes such as redness, edema, and necrosis. Movement of the treated joints was very restricted.
Estimation of Changes in the Circulating Immune Complex Systems. Platelet-rich plasma was obtained from the control rabbits by collecting their blood in citrate-coated tubes
and centrifuging at 600 X g for 10 minutes. Serum was obtained from control rabbits and those treated with either croton oil, leukocytes, or Freund's complete adjuvant. The rabbits with adjuvant arthritis received an intraperitoneal
injection of Freund's complete adjuvant 10-12 days before the
intraarticular injection. Serum from the rabbits with adjuvant
arthritis is known to contain immune complexes and therefore
served as a positive control. One milliliter of platelet-rich
plasma was mixed with 100 pl of serum and incubated at 37°C
for 10 minutes with occasional gentle shaking. The mixture
was centrifuged at 600 X g for 10 minutes. The supernatant
was diluted with saline and counted with a Coulter counter.
Decrease in the counts as compared to the control mixture was
considered a direct measure of platelet aggregation or formation of the immune complex.
Estimation of Activities of Various Lysosomal Enzymes
in Joint Synovial Fluid and Articular Cartilage. One milliliter of
sterile saline was injected into the intraarticular space of the
control and treated knee joints. The fluid was aspirated after
partial movement of the joint for a few seconds. The animals
were sacrificed and the articular cartilage was removed. The
cartilage was chopped into very fine pieces and homogenized
in saline in the presence of 0.1% Triton X-I00 in a Virtis
homogenizer.
Estimations of enzyme activities in the synovial fluid
and the cartilage homogenate were carried out as follows: 0glucuronidase activity was measured with p-nitrophenyl-0-dglucuronide as the substrate (16). Acid and alkaline phosphatase activities were estimated with p-nitrophenyl phosphate as
the substrate at pH 5 and pH 8 respectively (l6,17).
Tosyl-arginyl methyl ester served as a substrate for the estimation of trypsin activity (18). Cathepsin D activity was measured by the method of Ali et a1 (19) and collagenase by the
method of Nagai et a1 (20).
Synthesis of Collagen by Cartilage Explants in Vitro.
Freshly dissected articular cartilage (200-400 mg) from control and treated rabbit joints was chopped and incubated with
10 ml of Hanks' balance salt solution (Gibco), containing 100
pc 2,3-3H-proline (specific activity: 30-50 Ci/mmole), lo00 U
of penicillin, 1 mg of streptomycin, 50 pg/ml ascorbic acid,
and 50 rg/ml 8-aminopropionitrile for 16 hours at 37°C with
gentle shaking under an atmosphere of 95% 0,-5%CO,. At
the end of the incubation period the medium was removed; the
cartilage was washed with saline, chopped into fine pieces, and
extracted with 0.45 M NaCI, pH 7, overnight a t 4°C (21). The
extracts were dialyzed against 0.05 M Tris buffer, p H 7.5, and
separated into collagen and proteoglycan components on
DEAE cellulose column (0.9 X 30 cm) by elution with 0.2
M NaCl and then with 1 A4 NaCl in Tris buffer, p H 7.5. The
labeled collagen fraction was pooled, dialyzed against 0.06 M
Na acetate buffer, pH 4.8, containing 1 M urea, mixed with 2
mg of rat skin acid soluble collagen, which served as a carrier,
and chromatographed on C M cellulose column (0.9 X 10 cm)
at 40°C. The elution was carried out with the same buffer with
a linear gradient between 0 and 0.1 M NaC1. The effluent was
monitored at 230 mp and the distribution of radioactivity
under each peak was measured. The fractions corresponding
to each subunit of collagen were pooled and the distribution of
radioactivity between proline and hydroxyproline was estimated by the method of Rojkind and Gonzales (22) by using a
correction factor for 3H-proline.
A similar experiment was carried out using 100 pc of
U-"C lysine (specific activity > 270 mCi/mmole). The fractions corresponding to each subunit of collagen separated by
CM cellulose chromatography were pooled, dialyzed, and hydrolyzed. The distribution of radioactivity between lysine and
METABOLIC CHANGES DUE TO INFLAMMATION
20 1
A
Fig 1. Photomicrographs of the articular cartilage from croton oil-treated rabbit knee joints. A. AIcian
blue-PAS stain. B. Hematoxylin-eosin stain.
DESHMUKH AND HEMRICK
202
Table 1. Formation of Immune Complex by the Serum of Control
and Treated Rabbits
Percent Unaggregated
Platelets in Mixture*
Treatment
None
100
98
96
42
Croton oil
Peritoneal leukocytes
Freund's adjuvant
* Platelet-rich plasma
( I ml) and serum from control and treated
rabbits (100 pl) were incubated at 37°C for 10 minutes and centrifuged. Formation of immune complex was judged from the extent
of aggregation of platelets.
hydroxylysine residues was estimated with a Beckman amino
acid analyzer.
Studies on the Rate of Biosynthesis of the Matrix Components of Cartilage. 1) Diced articular cartilage of control and
inflamed joints in 100-mg batches were preincubated at 3 7 T
with 5 ml of Hanks' balanced salt solution containing penicillin-streptomycin, BAPN, and ascorbic acid under an atmosphere of 95% 0,-5% CO,. After 30 minutes one of the following isotopes was added: a) lOpc of %-Na,SO, (specific activity
>200 Ci/mmole) or b) 10 pc of 2,3-3H-proline. The
incubations were continued for various time periods. At the
end of each time period the medium was removed and cartilage
pieces were washed and hydrolyzed with 6 N HCl. The radio-
activity in the hydrolysates and the distribution of radioactivity into proline and hydroxyproline were estimated (22).
2) Cartilage explants were incubated for various time
periods as described above. The cartilage pieces were removed
and extracted with 0.45 M NaCl, pH 7, for 24 hours followed
by 4 M guanidinium chloride for 24 hours at 4°C. The dialyzable and nondialyzable radioactivity incorporated into each
extract were estimated.
3) The cartilage was preincubated in the medium for
30 minutes. Then 10 pc of 36S-Na2S04or 10 pc of 2,3-*Hproline were added to the medium and incubation was continued for 45 minutes. The radioactive medium was removed and
replaced by 10 ml of fresh medium containing 10 pM NaSO,
or proline. The chase of the label was carried out for various
time periods. The cartilage was hydrolyzed, the total radioactivity was incorporated into the hydrolysate, and the distribution of 3H-label in proline and hydroxyproline residues was
estimated as described earlier.
The total sulfate content of the cartilage was estimated
by using benzidine reagent (23); DNA content was determined
by the method of Giles and Myers (24) after extraction of
cartilage with 10% perchloric acid at 70" for 1 hour;
noncollagenous protein content was assessed by the Lowry
method (25); and collagen content was calculated by estimation of hydroxyproline (26). Total hexosamine and hexuronic
acid in the cartilage were also assayed (27,28). Moisture content was estimated from the total loss in weight of cartilage
after drying in a vacuum oven.
3H-thymidine uptake was estimated by incubating the
T
-
8a
Cathepsin
D
8-Glucuronidase
Acid
Alkaline
Trypsin
Phosphatase Phosphatase
Collagena se
Fig 2. Changes in the activities of various enzymes in the articular cartilage of normal and inflamed knee
joints. Clear bar = normal; hatched bar = treated with croton oil: dotted bar = treated with peritoneal
leukocyte Iysates.
METABOLIC CHANGES D U E TO INFLAMMATION
Cathepsin
D
B- Glucuronidase
Acid
Alkaline
Trypsin
Phasphatase Phorphatase
203
Collagenate
Fig 3. Changes in the activities of various lysosomal enzymes in the synovialj7uid of normal and injamed
rabbit knee joints. Clear bar = normal; hatched bar = treated with croton oil; dotted bar = treated wirh
peritoneal leukocyte lysates.
cartilage with 10 wc of ~nethyI-~H-thymidine
(40-60
Ci/mMole) for 30 minutes and then with 10 p M of unlabeled
thymidine for 4 hours. The cartilage was extracted with 10%
perchloric acid at 70°C and the radioactivity and total DNA
content of the extract were estimated (24).
RESULTS
Pathologic changes induced by intraarticular injections of croton oil or peritoneal leukocyte lysates
include swelling of the joint, increase in the amount of
synovial fluid, discoloration of the cartilage, and some
proliferation of synovial lining. All these changes were
more pronounced with the inflammation induced with
croton oil than with peritoneal leukocytes. The latter
part of the studies was therefore restricted to the inflammatory changes induced by croton oil.
Figures 1A and I B are photomicrographs of the
sections of articular cartilage from croton oil-treated
joint. In one experiment all the pieces of cartilage normally used for biochemical studies were subjected to
pathology studies. Serial sections were made from the
surface area to the deeper area of each piece. None of
the sections revealed any contamination with the fibrous
tissue, pannus, or osteophyte.
The capacity of the serum from normal and
treated rabbits to aggregate platelets is described in
Table 1. Whereas the serum from the rabbits receiving
Freund’s complete adjuvant shows appreciable formation of immune complexes, this phenomenon seems to
be virtually absent in the case of croton oil- or
leukocyte-treated rabbits, a finding suggesting that the
lesions produced are not related to circulating immune
complex formation. Nevertheless these results d o not
rule out the formation of immune complexes locally in
the joints.
The role of lysosomal enzymes as mediators of
inflammation and destruction of cartilage has been well
documented (6-10,29-3 1 ). Some of the lysosomal enzymes in the cartilage (Figure 2) and synovial fluid
(Figure 3) showed increased activity due to inflammation. The amounts of enzymes in the synovial fluid
and cartilage may vary according to the extent and
duration of inflammation. There was a definite increase
in the levels of acid phosphatase, cathepsin D, p-glucuronidase, and, in most of the experiments, in levels of
alkaline phosphatase in the synovial fluid as well as in
cartilage. Collagenase activity was higher in inflamed
joints, although not as marked as the activity of other
enzymes. N o change was observed in trypsin activity.
D E S H M U K H A N D HEMRICK
204
2
0.5
c
a1
0.4-
C
-800
;",
(?
I
cy
.-0
c
U
e
,
Y
4
L
0.2
-
10
30
20
40
SO
60
70
Effluent (ml)
A
-
2
m
0.5
0.4
1
-
- 800 .-
a1
C
0
,-\
i
It
N
c
U
\
-600
e
cc
-400
-200
10
20
30
Effluent
40
SO
60
70
(ml)
B
Fig 4. Chromatographic elution pattern of aH-proline-labeled collagen
from rabbit articular cartilage mixed with nonradioactive rat skin acid
soluble collagen on CM-cellulose column. Solid line = absorbance at 230
mp; broken line = elution of the labeled collagen. A. Normal cartilage.
B. Cartilage from the treated joint.
When control or inflamed cartilage was used as a substrate and exogenous trypsin was added to the reaction
mixture, or when exogenous trypsin was added to a
synthetic substrate in the presence of homogenized cartilage, trypsin activity was unchanged. This result rules
out the possible role of trypsin inhibitor in protecting
the cartilage from degradation due to the inflammation
induced by the agents under study.
Figure 4 illustrates the chromatographic elution
pattern of cartilage collagen on a CM cellulose column.
It is evident that 3H-proline-labeled collagen synthesized
by control cartilage elutes as one single peak where a1
chains of rat skin acid soluble collagen appear (Figure
4A), whereas collagen synthesized by the cartilage from
inflamed joints elutes as a1 and a2 peaks (Figure 4B).
Similar changes had been observed with human osteoarthritic cartilage earlier ( 5 ) , but the present data
seem to indicate that such alteration in the synthetic
pattern can occur merely by inducing the inflammation
by agents such as croton oil or leukocytes. Table 2
reveals the extent of such changes. The effect can be
reversed to some extent by discontinuing intraarticular
injections and allowing the rabbits to remain untreated
for 4-6 weeks.
The ratio of 3H-proline to SH-hydroxyproline in
the pooled fractions corresponding to a1and a2subunits
was typical for cartilage collagen.
The a1and a2chains synthesized by treated cartilage in presence of aH-lysine showed a distribution of
radioactivity different from that of normal chains. It is
known that CY chains of Type I collagen contain approximately 25-30 residues of lysine and 5-6 residues of
hydroxylysine, whereas al(II) chains of Type I1 collagen
from normal articular cartilage contain 15 residues o'f
lysine and 20-25 residues of hydroxylysine (32,33). Labeled a1chains from treated cartilage had a ratio of 3Hlysine: 3H-hydroxylysine = 2.0-2.9, a fact indicating that
there is a mixture of a1chains from Type I and Type I1
collagen. The a2 chains showed a typical ratio of lysine:hydroxylysine = 5.
Table 3 indicates that inflammation causes a significant depletion in the proteoglycan content of cartilage with a comparatively smaller decrease in collagen
content.
The data obtained with biosynthetic experiments
using isotopes shed light on the status of various components of the matrix. It can be seen from Table 4 that,
although the total amount of DNA in croton oil-treated
cartilage is slightly higher than in the control, uptake of
3H-thymidine or the specific activity is considerably increased and thus indicates an active proliferation of
chondrocytes.
Table 2. Effect of Inflammation on the Synthesis of Collagen by
Articular Cartilage in Vitro
Type
Control
Croton oil
Leukocytes
After 6 weeks*
Croton oil
Leukocytes
* These animals
q : a l Ratio
m
2.1
3.9
5.8
1.9
were given two injections and allowed to grow untreated for 6 weeks.
METABOLIC CHANGES D U E T O INFLAMMATION
205
Table 3. Biochemical Composition of Normal and Croton Oil-Treated Rabbit Articular Cartilage*
~
Moisture
Type
(% 1
Collagen
~
Noncollagenous
Protein
Uronic
Acid
Hexosamine
Sulfate
162.4 f 10.0
139.9 11.5
0.05
69.4 f 10.0
41.3 f 8.8
0.0I
51.1 f 6.0
39.0f 3.9
0.05
64.7 f 2.7
49.5 f 3.0
Normal
75.6 f 1.87 621.4f 27.1
Crotonoiltreated 77.5 f 1.5 574.8 + 51.1
0.1
0.1
ps
<0.001
* Values expressed as pg/mg
dry weight.
deviation.
$ Differences between the two groups are significant when P I0.05.For each estimation, n
t Standard
Figure 5 represents the total incorporation of
35S0, and SH-prolineinto the matrix components. The
synthesis of these components seems significantly enhanced in the croton oil-treated cartilage as compared
to normal. The incorporation of various labels in 0.45
M NaCl and 4 M guanidine extracts is shown in Figures
6A and 6B. A significant amount of radioactivity was
dialyzable in the case of guanidine extracts of croton
oil-treated cartilage. The data from the pulse-chase experiment (Table 5) indicate an active degradative process in the croton oil-treated cartilage. All these results
show that as a result of inflammation there is a rapid
breakdown and an active synthesis of matrix components. The increased rate of biosynthesis may be a
reflection of a repair mechanism.
DISCUSSION
Inflammation of the joints is one of the early
indications of articular involvement in rheumatoid arthritis and nonspecific synovial inflammatory states; it is
frequently associated with an osteoarthritic condition.
Inflammatory stimuli are either nonspecific or immunologically specific. The agents used to induce inflammation in these experiments seemed to have created no
detectable circulating immune changes. The inflammation was local in nature, as in the experiments in which
only one joint was treated, and the changes in the type
=
10.
of collagen synthesized were restricted only to the
articular cartilage in that particular joint.
Increased activity of lysosomal enzymes leading
to the degradation of cartilage matrix has been observed
in the synovial fluid and in the cartilage of joints involved in osteoarthritis and rheumatoid arthritis
(6-10,16,34-36). A collagenase active at neutral pH has
been isolated from human rheumatoid synovium (37).
Subsequent studies have shown that collagenase activity
is not limited to rheumatoid synovium but is also present in various joint diseases (38).
Higher levels of cathepsin D type enzyme, which
is active a t neutral pH and is specific for cartilage proteoglycans, have been observed in osteoarthritic human
cartilage (7-9). This enzyme seems to play a predominant role as a lysosomal protease in the degradation of cartilage matrix. Until now it was thought
that the role of these enzymes was to act on the
proteoglycans in the matrix and t o cause a loss of metachromasia. The authors' recent findings, however,
151
4oq
H3-Hypro
H3-Proline
Table 4. Incorporation of aH-Thymidine in the Cartilage
Explants in Vitro
Normal
Croton oil treated
Pt
3.27 f 0.10*
3.76 f 0.07
0.05
172f 7
491 f 16
<O.OOl
* Standard deviation.
t
n
Differences between the two groups are significant when P I 0.05;
= 10.
Incubation Time (hr.)
Incubation Time (hr.)
Fig 5. Incorporation of aH-proline and asso.in the cartilage explants
= normal
afrer incubaiion in uitro for various time periods. 0--0
cartilage; 0 - 4 = cartilage from inflamed joints.
D E S H M U K H A N D HEMRICK
206
Incubation Time Ihr.)
Incubation Time(hrd
Incubation Time
h.)
Fig 6A. Incorporation of 3H-proline and "SO, in the 0.45 M NaCI
extracts of the cartilage explants after incubation for various time
= normal cartilage; 0 - 4 = cartilage from inperiods. 0--0
gamed joints.
indicate that normal bovine articular cartilage changes
its pattern of collagen synthesis from Type I1 to
Type I when incubated with rat liver lysosomal enzymes
(21). Similar alterations can be seen with certain individual lysosomal enzymes or by incubation of the cartilage
in the presence of vitamin A, which is known to be a
lysosomal labilizer (39). The present findings suggest
that as a result of inflammation there is an increase in
0
ti3- Proline
GuCl Ext.
U
n
2
4
6
16
Incubation l i m e (hr.)
1
4
6
1
6
Incubation Time (hrd
Fig 6B. Incorporation of 3H-proline and "SO, in the 4M guanidine
chloride extracts of the cartilage explants after various incubation peri= normal cartilage: @-+
= cartilage from inflamed
ods. 0--0
joints.
the lysosomal enzymes, which in turn directs the chondrocytes to produce Type 1 collagen in addition to Type
11. With a greater degree of inflammation, chondrocytes
seem to synthesize exclusively Type 1 collagen. These
lysosomal enzymes, by modifying the environment
around the chondrocyte or by reacting with the cell
surface components, may drastically alter the cell behavior.
Although the collagen content of human osteoarthritic cartilage does not vary significantly from
that of normal tissue (1,2), the qualitative changes seen
in the type of collagen synthesized are substantiated by
an electron microscopic observation that the collagen
fibers were larger in diameter than those in the normal
caftilage (40). There was a considerable variation in the
distribution of fibers, especially in the surface zones.
Similar to the human osteoarthritic cartilage,
rabbit articular cartilage from the inflamed joints was
significantly depleted of the proteoglycan components,
with a small change in the collagen content. The metabolic studies showed that with the degradative process
there existed an active repair mechanism. The cells in the
inflamed tissue synthesized Type I collagen in significant
amounts, in addition to Type I1 collagen. The ratio of
a,:a2chains varied between 2.5 and 4.5 depending upon
the severity of inflammation.
The most important finding in this series was that
if the induction of inflammation was discontinued for
4-6 weeks, swelling of the joints subsided, lysosomal
enzyme levels were substantially decreased, and the cells
commenced to synthesize more Type I1 collagen. This
reversible phenomenon suggests two possibilities: a) The
chondrocytes contain two sets of genes. Under the normal circumstances production of Type I1 collagen occurs as a result of the expression of one set of genes.
With modifications of the environment the gene expression from the other set is more evident and is reflected
by the synthesis of Type I collagen. b) There may be two
populations of chondrocytes present in the cartilage,
one capable of producing Type I collagen, and the other,
Type I1 collagen. Under normal conditions Type I population may be dormant and become activated only
under stress conditions. Once the stress is removed Type
I1 population takes over the role of collagen synthesis. If
the stress persists for a longer duration, Type I
population may exist in a dedifferentiated form and
appear fibroblastic morphologically.
Schiltz et a1 (41) have observed that 5 bromodeoxyuridine causes changes in the normal phenotypic expression of growing chick embryo chondrocytes in cultures. The altered chondrocytes sup-
METABOLIC CHANGES DUE T O INFLAMMATION
207
Table 5. Change in 3H-Proline and 86S0,Incorporated into Cartilage Matrix with Incubation Time
Control*
Croton Oil Treated*
incubation
Time
(hours)
8H-proline
3H-hydroxyproline
3'S0,
2
4
16
14,310
13,790
12,100
3,375
3,224
2,500
3,120
2,900
2,500
3H-proline 3H-hydroxyproline
17,931
10,500
8,051
5.551
2, I68
1,593
35S0,
4,550
2,400
1,275
Cartilage pieces were incubated with labeled isotope for 45 minutes. The radioactive medium was replaced with the medium containing 10 pM of nonradioactive proline or sulfate and the label was chased
for various time periods. The loss of radioactivity indicates the degradative activity of the cartilage.
* Values expressed as dpm/100 mg cartilage.
press the synthesis of chondroitin sulfate and commence
to produce Type I collagen. Morphologically these altered cells resemble fibroblasts. Proximity to the surfaces of kidney or liver cells, their exudates, or the
altered chondrocytes interferes with the characteristic
synthetic activity of normal chondrocytes (42). Layman
et a1 (43) on the other hand reported that, although
articular cartilage explants from young rabbit joints
synthesized Type I1 collagen in vitro, chondrocytes from
the same cartilage, when grown in monolayer cultures,
produced Type I collagen. No addition of an exogenous
agent was necessary for such change.
If it is a prerequisite for chondrocytes to dedifferentiate into fibroblasts in order to synthesize Type I
collagen, the reversibility of such condition is doubtful.
It can be postulated that some of the chondrocytes may
be dedifferentiated as a result of inflammation and that,
when the stress is removed, the normal cells synthesize
Type I1 collagen more actively than the dedifferentiated
cells and thus produce a mixture of collagen with higher
al:
aZratio. Nevertheless this possibility seems remote in
view of the findings of Chacko et a1 (42) that in a mixture of normal and altered chondrocytes the latter type
interferes with the synthetic activity of normal cells.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Michael Schinitsky for
pathologic studies of cartilage and Dr. Stephen Krane for
helpful suggestions. The expert technical assistance of Mr.
Wayne Kline is also gratefully acknowledged.
REFERENCES
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2. Sokoloff L: The Biology of Degenerative Joint Disease.
Chicago, University of Chicago Press, 1969
3. Anderson CE, Ludowieg J, Harper HA, et al: The composition of the organic component of human articular
cartilage. J Bone Joint Surg 46A:I 176-1 183, 1964
4. Bollet AJ, Nance JL: Biochemical findings in normal and
osteoarthritic articular cartilage. I I . J Clin Invest
45:1170-1177, 1966
5. Nimni M, Deshmukh K: Differences in collagen metabolism between normal and osteoarthritic human articular
cartilage. Science 181:751-752, 1973
6. Weissmann G, Davies D T P A neutral, lysosomal protease active on protein polysaccharides and histones.
Arthritis Rheum 12:703, 1969 (abstr)
7. Sapolsky AI, Altman RD, Howell DS: Cathepsin D activity in normal and osteoarthritic human cartilage. Fed
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