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Influence of the severity and duration of murine antigen-induced arthritis on cartilage proteoglycan synthesis and chondrocyte death.

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813
INFLUENCE OF THE SEVERITY AND DURATION OF
MURINE ANTIGEN-INDUCED ARTHRITIS ON
CARTILAGE PROTEOGLYCAN SYNTHESIS AND
CHONDROCYTE DEATH
MARIJN W. M. KRUIJSEN, WIM B.
V A N DEN
Recent studies have shown that joint inflammation can suppress chondrocyte proteoglycan synthesis
and can even kill chondrocytes. In the present study, we
inveistigated the influence of the severity and chronicity
of niurine antigen-induced arthritis on the degree of
these toxic effects on chondrocytes. Joint inflammation,
quantitated by measurements of 99"'technetium pertechnetate uptake, was significantly correlated with the
inhibition of proteoglycan synthesis, measured by "Ssulfate incorporation. Histologic grading of the extent of
chondrocyte death on day 28 after arthritis induction
correlated best with the degree of inflammation present
on day 14. High scores for chondrocyte death were
found only in mice with persistent, severe arthritis. Our
data indicate that the severity of joint inflammation is a
major determinant of the degree of chondrocyte proteoglycian synthesis inhibition, whereas both the severity
and chronicity of the inflammation determine chondrocyte killing and, therefore, irreversible joint destruction.
Rheumatoid joint inflammation is characterized
by a protracted or chronic course and by fluctuations
in thle severity of the inflammation. The disease process often results in irreversible joint damage, but the
underlying pathogenic mechanisms that determine
__
-
From the Department of Rheumatology, University Hospital, St. Radboud, Nijmegen, The Netherlands.
Marijn W. M. Kruijsen, MD; Wim B. van den Berg, PhD;
Levinids B. A. van de Putte, MD: Professor of Rheumatology.
Address reprint requests to Dr. W. B. van den Berg,
Department of Rheumatology, University Hospital, St. Radboud,
6525 G A Nijmegen, The Netherlands.
Submitted for publication February 24, 1984; accepted in
revised form November 28, 1984.
Arthritis and Rheumatism, Vol. 28, No. 7 (July 1985)
BERG, and LEVINUS B. A.
V A N DE
PUTTE
joint damage remain largely unknown. Much attention
has been focused on the presumed enzymatic breakdown of cartilage (I), although the importance of this
mechanism in vivo is uncertain (2). Recent studies
have indicated that another mechanism might be important in cartilage destruction. Using antigen-induced
arthritis as an experimental model, we and other
researchers have shown that joint inflammation profoundly influences the functional and structural integrity of articular chondrocytes (3,4). This type of joint
inflammation was found to be capable of suppressing
proteoglycan synthesis and even killing chondrocytes.
We have found that the numbers of dead chondrocytes
increase on consecutive days after arthritis induction
(3). Studies on the influence of the severity and
chronicity of arthritis on these toxic effects on chondrocytes have not yet been reported.
Therefore, in the present study, we investigated
whether the severity and chronicity of murine antigeninduced arthritis determine the degree of toxic effects
on chondrocytes. These studies were technically possible because we recently developed reproducible and
sensitive methods of quantitating mouse knee joint
inflammation ( 5 ) and chondrocyte proteoglycan synthesis (6). The results indicate that the severity of the
joint inflammation is a major determinant of the degree
of inhibition of chondrocyte proteoglycan synthesis,
whereas both the severity and chronicity of inflammation determine the degree of chondrocyte killing and,
therefore, determine irreversible joint destruction.
MATERIALS AND METHODS
Animals and arthritis induction. Male CS7BL mice
were immunized, as described previously (3), by repeated
814
R/ L
KRUIJSEN ET AL
tio of 99mTc-uptake
0
2 .o
8
1.5
a
0
0
1.0
2
7
14
28
days after arthritis induction
Figure 1. Sequential measurements of yymTcuptake after arthritis
induction. Arthritis was elicited in the right knees of male C57BL
mice by intraarticular injection of either 0.6, 6, or 60 Fg of
methylated bovine serum albumin. Each point represents the *"Tc
uptake ratio (WL = right knee versus left knee) for 1 mouse. The
horizontal line shows the group mean value.
injections of methylated bovine serum albumin (mBSA;
Sigma, St. Louis, MO) emulsified in Freund's complete
adjuvant (Difco, Detroit, MI). Bordetella pertussis (National
Institute of Public Health, Bilthoven, The Netherlands) was
administered intraperitoneally as an additional adjuvant.
After 21 days, arthritis was induced by injecting
mBSA-saline into the right knee joint. To vary the severity
of inflammation, different doses of antigen-0.6, 6, or 60 pg
of mBSA in 6 pl saline-were administered intraarticularly
in several groups of mice. The left knee joints were injected
with 6 pl of saline.
99m
Tc uptake measurement. Joint inflammation was
determined by measurements of 99mTcpertechnetate uptake
in the knee joints, as described in detail elsewhere (5). One
of us (MWMK) performed all measurements, without knowledge of the group to which the mouse belonged. Briefly, 10
pCi of 9 9 m Tin~ 0.2 ml of saline was injected intraperitoneally
into the mice. After 10 minutes, the animals were sedated by
intraperitoneal administration of 4.5% chloral hydrate, 0.1
m1/10 gm of body weight. Then, at 20 minutes after injection
of %"'Tc, gamma radiation over the knee joints was measured using a scintillation crystal. A specially designed
device which fixed the knee joint into a well-shaped cavity
with a perspex window was used to hold the knee so that all
determinations were made at the same area of all knees and
at the same distance from the scintillation crystal. The rest of
the body of the mouse was covered with a lead shield (5).
Each knee joint was alternately counted 3 times (30
seconds per count), and the values were averaged. Uptake
values in normal joints ranged from 2,900-3,500 counts/30
seconds. Arthritis was scored as the ratio of the 9 9 m T ~
uptake in the right knee versus that in the left knee ( R : L
ratio of 9 9 m Tuptake).
~
R: L ratios of > 1.10 were considered
significant. In control experiments in which both knee joints
were injected with saline, R : L ratios of 9 9 m Tuptake
~
never
exceeded 1.10.
Incorporation of 35S into patellar cartilage. We used a
recently described method to determine 35S incorporation
(3,6). Briefly, patellae from arthritic joints and from control
joints were carefully removed, leaving a minimal area of
noncartlaginous tissue surrounding the cartilage. The patellae were placed in 5 ml of incubation medium consisting of
RPMI-HEPES and RPMI-HC03 (1 : 1) (Flow Laboratories,
Irvine, Scotland), with added penicillin (100 unitslml), streptomycin (100 pg/ml), and L-glutamine (2 mM). After a 45minute preincubation at 37°C in a 5% COz atmosphere, the
tissue specimens containing intact patellar cartilage were
transferred to 2-ml plastic incubation vials containing 0.5 ml
of RPMI and 5 pCi of 35S-sulfate (Radiochemical Centre,
Amersham, England), and incubation was continued for 3
hours. At the end of the incubation period, the patellae were
washed 3 times with 10 ml of saline, fixed overnight in 0.5%
cetylpyridinium chloride in 4% phosphate buffered formalin,
and decalcified in 5% formic acid for 1 day to enable proper
dissection of the patella from the surrounding connective
tissue.
After solubilization in 1 ml of Lumasolve containing
quaternary ammonium hydroxide (Lumac, Basel, Switzerland), the 35S radioactivity of the patellar cartilage was
counted in a liquid scintillation spectrometer. The decrease
in the 35S content of the right knee joint is expressed as a
percentage of the 35Scontent of the left knee joint of the
same animal and was calculated as follows:
1-
35S content of right patellar cartilage
x 100%
35Scontent of left patellar cartilage
Mice were selected for weight and age at the beginning of the experiment, to minimize variability in the absolute values of 35S incorporation (6). For all mice with
unilateral arthritis, the mean value of the 35Scontent of the
left control patella was 765 counts per minute (SD 154). No
significant differences in control values were found among
the various antigen-dose groups. The 35S content of the
arthritic cartilage ranged from 152-479 cpm (mean 297).
Histologic grading of inflammation and cartilage damage. Mice from different antigen-dose groups were killed by
ether anesthesia on day 28 after arthritis induction. The knee
joints were removed in toto and processed for histologic
examination (3). Serial sections ( 6 4 were made of whole
joints in a standardized frontal plane, including patella,
femur, and tibia. Some sections were stained with hematoxylin and eosin, and others were stained with alcian blue
(0.3M MgC12, pH 5.4).
PROTEOGLYCAN SYNTHESIS AND CHONDROCYTE DEATH
Oh
decrease of
35scontent
.*
,1
*
*
A
* A
I
1.20
1.40
1.60
R/L ratio of Tc uptake
Figure 2. Correlation between the E L (right knee versus left knee)
ratio of wmTC uptake and the percentage decrease of ''S
incorporation into arthritic cartilage of individual mice on day 2 after
arthritis induction. Arthritis was elicited in the right knee with either
0.6 pg (O), 6 pg (A), or 60 pg (*) of methylated bovine serum
albumin. The correlation coefficient, r = 0.53, indicated a significant
correlation, P < 0.05 (n = 17).
815
severity of joint inflammation on 35S-sulfate incorporation into anatomically intact mouse patellae was
measured on day 2 after arthritis induction. Figure 2
shows that in all instances, even in animals with
relatively mild inflammation, a decrease in "S-sulfate
incorporation was found. We also found a significant
correlation between the degree of inflammation and
the percent of decrease in 35S-sulfate incorporation
(Figure 2).
To investigate whether the inhibition of 35Ssulfate incorporation was due to the inflammation
rather than to a toxic effect of the injected antigen, 6
pg of mBSA was injected into the right knee joints of
nonimmunized mice. This dosage of antigen did not
induce inflammation, and no decrease in "S-sulfate
incorporation was found (mean value -8%, SD 20). In
addition, we investigated whether the mere trauma of
intraarticular injection in immunized animals causes
suppression of 35S-sulfate incorporation, by injecting 6
pI of saline into the right knees of immunized mice.
We found no signs of inflammation and no decrease in
35S-sulfate incorporation (mean value 2%, SD 4).
Influence of arthritis severity on chondrocyte
death and cartilage damage. We studied the association
of parameters of joint inflammation with the degree of
resulting chondrocyte death (Figure 3) and the loss of
The arthritis score was determined by grading the
infiltrate and the exudate, with the examiner having no
knowledge of the group from which the specimen was obtained. The extent of cellular infiltration of the synovial tissue
was graded 0-3 according to the total number of inflammatory
cells present. The cellular exudate in the joint space was also
graded 0-3. Loss of chondrocytes (chondrocyte death score)
in patellar cartilage was graded 0-3, where 0 = no loss of
chondrocytes and 3 = loss of all chondrocytes. Loss of
alcian blue staining of the arthritic cartilage, which reflects
decreased proteoglycan content (7), was scored by comparison with the contralateral control joint of the same animal:
0 = similar staining, 1 = slightly decreased staining, 2 =
definitely decreased staining, 3 = severely decreased staining. The final score for each animal was the mean of the
scores on 4 sections taken from a standard area of the joint.
RESULTS
To induce different degrees of knee joint inflammation, groups of mice were injected intraarticularly
with different doses of antigen, i.e., 0.6, 6, or 60 p g of
mBSA. As a result, the severity of inflammation
varied widely, as demonstrated by the R:L ratios of
technetium uptake, both in the acute and chronic
phases of arthritis (Figure 1).
Influence of severity of joint inflammation on
chonrdrocyte 35S-sulfateincorporation. The effect of the
Figure 3. Knee joint section of C57BL mouse, showing the patella
(P)and the opposite femur (F) on day 28 after elicitation of antigeninduced arthritis. The cartilage (C) shows numerous empty lacunae,
indicating chondrocyte death (hematoxylin and eosin stained, magnification x 250).
816
KRUIJSEN ET AL
Figure 4. Alcian blue staining of knee joint sections of C57BL mouse, showing the patella (P)and opposite femur (F). A , Normal joint: B,
Arthritic ioint on dav 28 after arthritis induction. Note the decreased staining of cartilage and the presence of exudate cells in the joint space.
(Magnification x 200.)
alcian blue staining on day 28 (Figure 4). R : L ratios of
technetium uptake on days 2 , 7 , 14, and 28 all showed
significant correlations with scores for chondrocyte
death (Figure 5 and Table 1) and scores for the loss of
alcian blue staining (Table 1).
We also correlated histologic exudate and infiltrate scores obtained on day 28 with the day 28 scores
for chondrocyte death and loss of alcian blue staining
(Table 1). These correlation coefficients were even
higher than those for day 28 R : L ratios of technetium
uptake, correlated with either the chondrocyte death
score or the score for the loss of alcian blue staining.
This probably reflects the fact that R : L ratios on day
28 are determined not only by inflammatory changes,
but also by proliferative changes in the joint.
We attempted to determine the relative importance of the severity of acute and chronic joint inflammation to the resulting degree of chondrocyte death.
For this purpose, we classified acute (day 2) and
Table 1. Correlation coefficients (r) between measures of the
severity of inflammation and those of cartilage damage on day 28
after arthritis induction*
CHONDROCVTE D E A T H
Figure 5. Correlation between chondrocyte death and ""T
'c
uptake
after arthritis induction. A significant correlation, P 5 0.001, is
reached at r = 0.67 (n = 21). The highest correlation was reached on
'c
uptake in the right knee
day 14. R/L ratio = the amount of ""T
versus that in the left.
R:L ratio of ""T
'c
uptake
Day 2
Day 7
Day 14
Day 28
Exudate in joint cavity, day 28
Infiltrate in synoviurn, day 28
Chondrocyte
death
Loss of
alcian blue
staining
0.67
0.74
0.84
0.73
0.90
0.85
0.77
0.70
0.77
0.61
0.85
0.89
* R:L ratio = uptake value in the right knee versus that in the left.
See Materials and Methods for scoring systems. A significant
correlation, P 5 0.001, is reached at r = 0.67 (n = 21).
PROTEOGLYCAN SYNTHESIS AND CHONDROCYTE DEATH
Table 2. Classification of mice according to histologic score for
chondrocyte death (day 28) and severity of previous inflammation*
R:L ratio of 99"Tc
uptake
Number of mice
showing chondrocyte death
Group
Day 2
Day 14
<1.35
>1.35
1
2
<1.61
>1.61
c1.61
>1.61
c1.13
c1.13
>1.13
>1.13
8
3
0
0
I
4
0
5
3
4
* R:I, ratio = uptake value in the right knee versus that in the left.
The discriminating points are the mean of the values obtained for
that parameter in all mice. (See also Figure 1.)
chronic (day 14) joint inflammation (measured by R : L
ratios of technetium uptake) as either mild or severe
and chondrocyte death scores (day 28) as either high
or low, taking the mean value of these parameters as
disciriminating values.
Table 2 shows that severe acute and severe
chronic inflammation (group 4) resulted in high chondroc yte death scores in all instances, whereas mild
acute and mild chronic inflammation (group 1) was
always associated with low chondrocyte death scores.
Interestingly, all 3 mice with severe acute and mild
chronic inflammation (group 2) had low chondrocyte
death scores, whereas 4 of 5 mice with mild acute and
severe chronic arthritis (group 3) had high chondrocyte death scores. This suggests that chronicity of
arthritis is an important determinant of the ultimate
extent of chondrocyte death.
DISCUSSION
Our data indicate that antigen-induced joint
inflammation suppresses chondrocyte function even
when the inflammatory process is relatively mild.
Suppression of chondrocyte proteoglycan synthesis
was shown to be clearly dependent on the severity of
the joint inflammation. Chondrocyte killing, and therefore irreversible damage to the cartilage, was determined by both the severity and the duration ofjoint
inflammation.
The severity of antigen-induced arthritis in mice
appetars to be dependent on the dose of intraarticular
antigen, both in the initial phase and in the chronic
phase. We have found that 60 pg of mBSA usually
induces severe inflammation, 0.6 pg of mBSA always
causes mild inflammation, and 6 pg of mBSA produces
a variable response. The dose response probably follows a sigmoidal curve, which shifts according to the
degree of immunity to the antigen. Small differences in
817
delayed hypersensitivity, which may occur after seemingly identical immunization protocols, may profoundly influence the severity of the joint inflammation that
is induced with the intermediate antigen dose.
Recent studies have shown that in experimental
arthritis, including antigen-, carrageenin-, and zymosan-induced arthritis (3,4,8), chondrocyte synthetic
function is suppressed. These studies have been performed both in vitro and in vivo, using 35S-sulfate and
3H-acetate as labeled precursors. Mechanisms involving in vivo suppression of this function are unknown,
but in vitro studies have indicated that there are
several potential mediators. It has been shown that
lymphokines (9), prostaglandins (10,l l ) , and humoral
factors from synovial tissue (12) can suppress chondrocyte function. These factors may be released from
inflammatory cells and synovial cells, all of which are
present in increased numbers during the inflammatory
process (13). In addition, local oxygen tension, which
has been shown to be decreased in inflamed joints (14),
may have a modulating effect on chondrocyte proteoglycan synthesis (15). Moreover, since nutrients reach
the chondrocytes via the synovial fluid, the exudate in
the cavity of the inflamed joint may interfere with the
nutrient supply to the chondrocytes.
In vivo studies using antigen-, zymosan-, and
carrageenin-induced arthritis have also indicated that
chondrocyte suppression is reversible (3,4,8). In vitro
studies have shown reversibility of chondrocyte suppression for all of the above-mentioned mediators
except prostaglandins (10). Our studies indicate that,
at least in the early phase of inflammation, the degree
of inhibition of chondrocyte proteoglycan synthesis
depends on the severity of inflammation. This dependency has not been previously reported, probably
because of technical difficulties in quantitating the
inflammatory process. However, in vitro studies have
reported dose-dependent chondrocyte suppression for
lymphokines (9) and, recently, for oxygen metabolites
(16).
Recent studies have noted chondrocyte killing
during antigen-induced and carrageenin-induced arthritis (3,4,8,17). Since this type of damage, unlike the
inhibition of proteoglycan synthesis, is irreversible, it
may be an important factor in joint destruction. Factors responsible for chondrocyte killing are, at present, unknown. Our data indicate that both the severity
and the duration of arthritis are important determinants of the phenomenon. Chondrocyte death correlated best with a late phase of arthritis (day 14),
indicating that chronicity of inflammation is an important determinant; moreover, high chondrocyte death
818
scores were found only in mice with persistent, severe
arthritis (Table 2).
Ultrastructural studies of cartilage from New
Zealand white rabbits with antigen-induced arthritis
have shown that considerable chondrocyte damage
can be seen in the early phase af inflammation (18).
How much damage can occur before these changes are
irreversible is unknown. Our data suggest that the time
factor may be important. One possible explanation
may be that chondrocytes become increasingly vulnerable as the inflammatory process continues over time.
In addition, chondrocytes are protected by the surrounding matrix in the early inflammatory phase,
allowing only small molecular weight mediators, such
as superoxide and hydrogen peroxide, to penetrate. In
the later phase, when severe proteoglycan depletion
has occurred, high molecular weight mediators, such
as lymphotoxin (19) or cross-reacting antibodies (20),
may also penetrate, leading to further damage to these
cells.
Apart from those factors involved in the inflammatory process, chondrocyte killing may be related to
other factors. Lowther and colleagues (4) have
stressed the importance of biomechanical factors,
since cartilage damage is found mainly in the loadbearing areas of the joint. Previous data obtained by
our group (3) point to another possible factor. In our
studies on murine patellar cartilage, dying chondrocytes were usually first seen in the central part of the
patella, suggesting that chondrocytes that are located
farthest from nutrient blood vessels are most vulnerable. Finally, local retention of antigen in the form of
immune complexes at special sites of the joint (21,22)
may determine the distribution pattern of chondrocyte
death. However, the role of immune complexes in
local damage, e.g., by complement activation, remains
a moot point.
Data on the influence of human joint inflammation on chondrocytes of intact hyaline cartilage are
presently unavailable; however, light microscopic and
ultrastructural pictures of this area have been published. These pictures of rheumatoid articular cartilage
show changes which suggest that chondrocyte killing
occurs, at least in the presence of rheumatoid arthritis
(23-27). If, according to our experimental data, mild
inflammatory processes suppress chondrocyte proteoglycan synthesis, then it is worthwhile for physicians
to attentively treat even a mild degree of joint inflammation. It is possible that cartilage integrity could then
be restored before irreversible damage has occurred.
KRUIJSEN ET AL
ACKNOWLEDGMENTS
The authors thank Marianne van der Sluis-van der
Pol, Wil A. Zwarts, and Wim J. M. van den Broek for their
technical assistance; P. B. Spaan, G. J. F. Grutters, and the
staff of the animal laboratory; and Liduine van den Bersselaar and Marlie Pierik for their secretarial assistance.
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