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The superficial layer of human articular cartilage is more susceptible to interleukin-1induced damage than the deeper layers.

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ARTHRITIS & RHEUMATISM
Vol. 39, No. 3, March 1996, pp 478-488
0 19%, American College of Rheumatology
478
THE SUPERFICIAL LAYER OF HUMAN ARTICULAR CARTILAGE IS
MORE SUSCEPTIBLE TO INTERLEUKIN-1-INDUCED DAMAGE THAN
THE DEEPER LAYERS
H. J. HAUSELMANN, J. FLECHTENMACHER, L. MICHAL, E. J.-M. A. THONAR, M. SHINMEI,
K. E. KUElTNER, and M. B. AYDELOTTE
Objective. To compare the responses of chondrocytes from superficial and deep layers of normal human
articular cartilage to interleukin-1 (IL-1) and IL-1 receptor antagonist protein (IRAP), and to evaluate the
binding sites for IL-1 on these cells.
Methods. Cartilage and chondrocytes from superficial and deeper layers of human femoral condyles
were cultured with and without IL-1 in the presence and
absence of IRAP. The effect of these agents on %proteoglycan synthesis and catabolism and production
of stromelysin and tissue inhibitor of metalloproteinases
1 (TIMP-1) were measured by biochemical and immunologic assays. Receptor binding was evaluated using
‘251-labeledIL-1.
Results. IL-1 induced more severe inhibition of
proteoglycan synthesis and a lower ratio of secreted
TIMP-kstromelysin in chondrocytes from superficial
cartilage than those from deeper cartilage. IRAP
blocked responses to IL-1 more effectively in chondroDr. Hauselmann’s work was supported by Swiss National
Science Foundation grant 3 1-33791.92, a Swiss Fellowship from the
Bernardi Foundation, University of Bern, and by the Hemnann
Foundation, Vaduz, Liechtenstein. Dr. Thonar’s work was supported by NIH grant AG-04736. Dr. Kuettner’s and Dr. Aydelotte’s
work was supported by NIH grant AR-39239 and by an Arthritis
Research Grant from Hoechst A-G, Werk Kalle-Albert, Germany.
H. J. Hauselmann, MD: Rush Medical College at RushPresbyterian-St. Luke’s Medical Center, Chicago, Illinois, the
University Hospital, Zurich, Switzerland, and the University of
Berne, Berne, Switzerland; J. Flechtenmacher, MD, Rush Medical
College at Rush-Presbyterian-St. Luke’s Medical Center, and the
University of Ulm, Ulm, Germany; L. Michal, BS, E. J-M. A.
Thonar, PhD, K. E. Kuettner, PhD, M. B. Aydelotte, PhD: Rush
Medical College at Rush-Presbyterian-St
Luke’s Medical Center;
M. Shinmei, MD, PhD: National Defense Medical College, SaitamaKen, Japan.
Address reprint requests to H. J. Hauselmann, MD, Department of Rheumatology, University Hospital, 8091 Zdrich, Switzerland.
Submitted for publication April 10, 1995; accepted in revised form September 22, 1995.
.
cytes from deep cartilage than those from superficial
cartilage. Chondrocytes from the articular surface
showed approximately twice the number of h i g h - m i t y
binding sites for IL-1 as did cells from deep cartilage.
Conclusion. Chondmytes from the surface of articular cartilage show a greater vulnerability to the harmful effects of IL-1 and are less responsive to the potential
therapeutic effects of IRAP than cells in the deeper layers
of the tissue.
Treatment of cartilage tissue in vitro with IL-1
(1,2) or injection of IL-1 into the synovial cavity (3,4)
results in inhibition of synthesis of proteoglycans
(PGs) (5-9), enhanced production of prostaglandin E2
(PGE,) (10) and metalloproteinases (1 l), and excessive
catabolism of PGs. Together these conditions contribute to loss of cartilage matrix and inadequate tissue
repair. Based on experimental results and on the
finding of IL-1, PG fragments, and proteolytic enzymes in inflamed joints (12-14), it is widely accepted
that IL-1 plays an important role in cartilage erosion in
inflammatory joint diseases. Because PG synthesis is
usually upregulated during the early stages of osteoarthritis (15), IL-1 has been given little attention as a
potential mediator of early metabolic changes in articular cartilage. In patients, on the other hand, especially during the inflammatory episodes which almost
always develop at late stages of the disease, IL-1
appears to play a significant role in suppressing the
synthesis and repair mechanism (16). IL-1 a and IL-Ip,
the 2 agonist isoforms of 1L-1 (17-21), bind to specific
cell-membrane receptors and set in motion complex
signal transduction pathways which are not yet fully
understood (22). The human recombinant form of IL-1
receptor antagonist protein also binds to the same
receptors, but since it fails to initiate signal transduction or elicit biological responses, it blocks cellular
ARTICULAR SURFACE SUSCEPTIBILITY TO IL-1
responses to IL-1 (23) and acts as a pure antagonist
(24-28).
Two structurally related cell membrane receptors for IL-1 have been identified, the 80-kd type I
receptor and the 68-kd type I1 receptor (29-32). The
type I receptor occurs on all cells that are responsive
to IL-I, including human chondrocytes (16,33), while
the type I1 receptor occurs on B cells, monocytes,
neutrophils, and bone marrow cells (34). However,
some cells can express both types of receptor, a
feature which may increase the range of responses
elicited by IL-1 (34,35). The coexpression of type I and
type I1 receptors for IL-1 has also recently been
reported in human synoviocytes (36). The smaller type
I1 receptor has a shorter cytoplasmic domain
(31,34,37,38). Apparently, it is not a signalling receptor, but acts as a “decoy target” at the cell surface or
extracellularly in soluble form, trapping IL-I and
thereby diminishing the effective signalling of IL-1 via
its type I receptor (39). Although IL-la and p bind to
the same receptors, IL-1a seems to bind preferentially
to the 80-kd type I IL-1R (40) and IL-Ip binds best to
the 68-kd type I1 IL-1R (41). IRAP reportedly binds
well to the membrane-bound type I IL-1R and soluble
murine type I1 IL-lR, but only poorly to membranebound type I1 IL-1R of murine p r e B cells (42). IRAP
can inhibit the binding of both IL-la and p to the
type I IL-1R on T cells and to the type I1 IL-1R on
polymorphonuclear leukocytes and Raji human B
lymphoma cells (26,43).
Earlier studies of cartilage organ cultures and
chondrocyte cultures were valuable for evaluating the
direct effects and mechanisms of action of modulators
such as IL-1 (5-9). In those studies, the effects of IL-1
were examined on the entire thickness of cartilage (or
chondrocytes therefrom), without consideration of the
heterogeneity of articular cartilage. However, articular cartilage is a highly complex, stratified tissue with
depth-related differences in biochemical composition,
macromolecular organization, and biomechanical
properties, which result ultimately from metabolic
specialization among the resident chondrocytes (for
review, see ref. 44). In the arthritides, the distribution
of lesions and the rate of their progression are related
to this heterogeneity within the tissue. For example, in
osteoarthritis, the earliest damage is detected in the
most highly cellular, thin superficial zone (45). In
addition, even in healthy joints, the progressive thinning of articular cartilage with age appears to be
mainly at the expense of the deeper layers.
We have previously demonstrated that in cul-
479
ture, tissue slices and subpopulations of articular
chondrocytes isolated from dfierent layers of cartilage
continue to express metabolic features characteristic
of their specific anatomical origins (46,47). Preliminary
experiments showed that in culture, both human and
bovine chondrocytes in specific layers of articular
cartilage respond in distinctly different, but characteristic, ways to treatment with IL-la, IL-lp, and IRAP
(48-50). Results of more detailed studies are reported
here, and show that in comparison with cells from
deep layers of human articular cartilage, the chondrocytes closest to the synovial cavity are more responsive to IL-1, are less readily protected by the naturally
occurring inhibitor, IRAP, and possess more receptors
for 1L-1. In addition, we present evidence that there
are clear differences between the effectiveness of the 2
isoforms of IL-1 in inhibiting PG synthesis by human
chondrocytes. To our knowledge, this is the first
report of affinity studies between ligand and receptors
performed in chondrocytes isolated from different
layers of cartilage and examined in suspension culture,
under conditions in which the normal cellular phenotype is conserved (51).
SUBJECTS AND METHODS
Chondrocyte and cartilage cultures. Tissues were
obtained from 30 femoral condyles (15 donors), 4 ankles (2
donors), and 1 shoulder (1 donor) from donors who had no
history of joint disease and had macroscopically normal joint
cartilage. Tissue was obtained through the Regional Organ
Bank of Illinois and through the Examiners Office of the
University of Bern, according to the institutions’ protocols
and with institutional approvals. Sixteen of the donors were
male (ages 14, 17, 22, 24, 29, 32, 37, 42 [2 donors], 50, 51,55
[2 donors], 57, and 63 [2 donors]) and 2 were female (ages 13
and 43).
For some experiments, articular cartilage representing the entire thickness of uncalcified tissue was cut from the
weight-bearing surfaces of the medial and lateral femoral
condyles. For most experiments, in order to obtain tissue
representing different layers of articular cartilage, very thin
superficial slices were collected separately from tissue that
was subsequently harvested from the combined middle and
deep layers of articular cartilage of the medial and lateral
femoral condyles. These 2 pools of tissue were rinsed,
blotted, and weighed to determine the ratio of wet weights.
For most of the experiments, the chondrocytes were
isolated by protease digestion and cultured in agarose gels
(46). Results were confirmed by culturing intact cartilage
slices. For binding studies, to evaluate IL-lR, chondrocytes
were cultured in alginate, another supporting gel in which
chondrocytes show normal phenotype expression (52), but
from which the cells can be released readily with chelating
agents and recovered for measurement of cell-bound radioactivity (52). Cultures were fed daily with Ham’s F-12
HAUSELMANN ET AL
480
medium (or with F-12DME for alginate cultures) supplemented with 10% pooled adult human serum and 25 &ml
ascorbate, and incubated at 37°C in a humidified atmosphere
of 5% CO, in air.
Treatment with cytokines. After maintenance for 4-6
days in control medium, triplicate or quadruplicate groups of
cultures were treated with IL-1a or IL-1p (human recombinant form; Genzyme, Boston, MA) at concentrations of
0.001-2.0 ng/ml. Other cultures were treated simultaneously
with IL-1 and IRAP (1-500 ng/ml) (human recombinant form
of IRAP kindly donated by Dr. Robert Smith, The Upjohn
Company, Kalamazoo, MI). Cytokines were added with the
medium each day to maintain the desired concentrations.
Measurement of the synthesis and catabolism of PG.
To assess PG synthesis, cultures were treated with cytokines
for 3 days, and then incubated for 4 hours with 20 &i/ml of
"S-sulfate (specific activity 2540 Ci/mg; Amersham, Arlington Heights, IL). PGs were extracted from the tissue
slices and agarose gel cultures under dissociative conditions
in 4M guanidinium chloride containing protease inhibitors,
as described previously (47). Samples were stored at -70°C
until analyzed.
'%-labeled PGs in the media and dissociative extracts were quantified by liquid scintillation spectroscopy
after chromatography on Sephadex G-25M in PD-10 columns, as described previously (47). Parallel cultures were
digested overnight at 60°C with papain, and the DNA content was measured by fluorescence using Hoechst dye 33258
(53). To measure the effect of IL-1 on PG catabolism,
chondrocytes in the agarose cultures were labeled overnight
in the absence of IL-I, then rinsed thoroughly and chased for
7 days with and without IL-I a. The rate of loss of '%-labeled
PGs from the agarose gel was calculated daily by measuring
the 3SS-labeledPGs appearing in the medium, as described
previously (4732).
Binding studies to evaluate IL-1 receptors. Receptors
for IL-I in human chondrocytes from superficial and deep
layers of articular cartilage were evaluated in direct-binding
assays according to published methods (5435). The following modification was adopted: before and during the binding
study, the cells were maintained in a 3-dimensional culture
system within alginate gels (52). After 4 days of culture in
control medium, alginate beads containing 4 0 , ~ 5 0 , 0 0 0
chondrocytes were distributed into %-well plates (1 bead
well) and incubated for 4 hours at 22°C in binding buffer, in
the presence of '251-labeledIL-la or IL-1p (New England
Nuclear, Boston, MA) up to a concentration of 36 ng/ml,
with triplicate samples for each concentration of IL-1. The
beads were washed extensively, and the cells were released
from alginate with 5 mM EDTA, recovered by centrifugation
or filtration, and then bound radioactivity was measured in a
gamma counter.
Specific binding was calculated as the difference in
binding of '251-labeledIL-I a or p alone and in the presence
of a 250-fold concentration of unlabeled IL-1a or p (54). The
molar ratios of '"I to IL-la and to IL-1p were the same. To
correct for differences in cell numbers, parallel cultures of
alginate beads incubated with unlabeled IL-la or p were
analyzed for DNA content (53). Results were calculated
manually and controlled with an appropriate computer program (Scatchard analysis; Biosoft, Cambridge, UK).
v
0,001
0,Ol
0,1
1
hrlL-la (ng/ml)
Figure 1. Synthesis of 35S-proteoglycans (35S-PGs) by cultured
articular chondrocytes. Chondrocytes from full-thickness cartilage
of 2 femoral condyles, 1 from a 22-year-old male (0)and 1 from a
63-year-old male (0).
were cultured in agarose gel and treated for 3
days with human recombinant interleukin-l a (hrIL- I a) at the concentrations shown. Note the dose-related inhibition of PG synthesis
in response to treatment with IL-la. Results were corrected for
DNA content and are expressed as percentages of "S-PG synthesis
in untreated control cultures. Bars show the SD of the mean (n = 4).
Similar results from additional experiments are shown in Table 1.
Assays for stromelysin, tissue inhibitor of metalloproteinases 1 (TIMP-I), and PGE,. Medium was harvested after
each day of treatment of cartilage slices; TIMP-1 and stromelysin were measured by enzyme-linked immunosorbent assay
(56,57). The assay system for stromelysin (MMP-3) measures
not only proMMP-3, but also the active form of MMP-3, as well
as MMP-3 complexed with TIMP-I or TIMP-2. The assay
system for TIMP-I is capable of measuring both free TMP-1
and TIMP-I complexed with MMP-I (collagenase). PGE, was
measured by a specific, commercially available radioimmunoassay (Cayman, Ann Arbor, MI). Results are shown for media
samples on the second day of treatment.
RESULTS
Effects of IL-1 on the entire mixed population of
human chondrocytes. Human chondrocytes isolated
from healthy knee cartilage and cultured in agarose
gels showed a dose-dependent inhibition of PG synthesis in response to IL-la (Figure 1). Interestingly,
the age of the donor appeared to play an important role
in the metabolic response; chondrocytes and cartilage
slices from the younger donors (ages 22 and 29)
showed similar inhibition of PG synthesis with lower
concentrations of 1L-1a (Table 1 and Figure 1) than did
cells from the older donors (ages 42, 55, and 63).
Because of the small numbers of young donors, a
statistical analysis between the average concentrations
of IL-1 a required to inhibit 35Sincorporation into PGs
ARTICULAR SURFACE SUSCEPTIBILITY TO IL-1
Table 1. Mean concentrations of interleukin-la (IL-la) required
to inhibit incorporation of 35S into proteoglycans by 50% (I(&) in
cultured slices of human articular catilage from the knees of 2 young
and 4 older donors*
Donor age
42
55
63
63
Mean 2 SD age
56 f 10
IC, IL- I a
(pdml)
Donor age
IC, IL- 1 a
(Pdml)
7.5
9.0
9.0
15
22
29
2.5
3.0
10.1 2 3.3t
26
2.75
120
glx 100
$
80
0
0
g
60
s 40
4 20
"
0
v)
* Results corrected for cell number (DNA content).
t Mean
48 1
* SD.
by 50% in young and old donors was not performed
(Table 1). The results were corrected for the cell
number (DNA content) and, in Figure 1, are expressed
as a percentage of the incorporation in untreated
control cultures of isolated chondrocytes. Very little
catabolic response was observed in human chondrocytes; over a period of 7 days, IL-la at 0.2 ng/ml and
2 ng/ml stimulated the loss of PGs by only -10% (data
not shown).
Differential responses to IL-1 by subpopulations
of chondrocytes. When populations of chondrocytes
from different layers of human cartilage were compared, it was evident that chondrocytes from the
superficial layer were much more responsive to both
isoforms of IL-1 at low concentrations than were those
from deeper layers of articular cartilage (Figure 2).
Interestingly, however, in both cell populations, PG
synthesis was inhibited to a greater extent by IL-1p
than by IL-la (Figure 2). Similar results with respect
to differences between isolated superficial and deep
chondrocytes in response to IL-la and p were observed in 2 other experiments using knee joint cartilage from 2 additional human organ donors ages 24 and
51 (data not shown). In addition, in slices of human
articular cartilage (2 knee joints, 1 ankle joint) from 3
different donors ages 13,37, and 51 years, similar differences in inhibition of PG synthesis were observed in
response to treatment with IL-la and p (Table 2).
The results in both cell and cartilage culture
were comparable, and illustrate the pronounced sensitivity of the superficial cartilage to IL-I, and the
greater responsiveness of human chondrocytes to
IL-I p than to the same concentration of IL-1a.Human
chondrocytes from either superficial or deep layers of
cartilage showed little catabolic response to IL-I (data
not shown).
0,Ol
0,1
h rlL-1a/p (ng/ml)
1
Figure 2. Comparisons of the inhibitory effects of treatment for 3
days with human recombinant interleukin-1a and p (hrIL-1alp;
opedsolid symbols) on the synthesis of 35S-proteoglycans(35S-PGs)
by articular chondrocytes derived from superficial (circles) and deep
(squares) zones of the femoral condyles of a 13-year-old female.
Knee chondrocytes from the articular surface showed relatively
greater inhibition than those from deep cartilage, and PG synthesis
was suppressed more by treatment with IL-Ip than with IL-la.
Results were corrected for DNA content, and are expressed as
percentages of the radioactivity in untreated control cultures. Bars
show the SD of the mean (n = 3). These results are representative of
3 experiments on cartilage from 3 different donors (see Table 2).
Responses to IL-1 in the presence of I M P .
Chondrocytes derived from different zones of human
cartilage also responded differently to simultaneous
treatment with IL-I a and IRAP. In chondrocytes from
the deeper layers of cartilage, the IL-1 *induced inhiTable 2. Effect of interleukin-1 a (IL-1a)and IL-Ip (0.5 n d d ) on
the incorporation of 35S into proteoglycans in cultured slices of
cartilage from 3 donors of different ages*
Superficial cartilage
Donor age
13
37
51
Mean f SD age
39.8 2 14.8
Deep cartilage
Donor age
13
37
51
Mean 2 SD age
39.8 2 14.8
IL- 1a
IL-IP
11.4
18.8
11.35
4.2
9.0
13.6
?
3.8ti
5.21
6.5
?
2.2t§
55.8
90.0
55.85
14
40.0
14.0
67.2 2 19.7t
22.7 f IS.Ot§
* Results corrected for cell number (DNA content). Values are
percentages of the incorporation in untreated control slices.
t Mean -e SD.
t P 5 0.05 versus deep cartilage.
5 P I0.05 versus IL-I a.
HAUSELMANN ET AL
482
120 I
120
100
80
60
40
20
IL-I(nghl)
0
0
0.5
0.5
0.5
0.5
IRAP(nglm1)
0
500
0
5
50
500
I-
Control
IL-la
IL-10 IRAP IL-la IL-10
+ I M P +IMP
B
A
F
m 3. A, Chondrocytes from the superficial and deeper layers of femoral condyles (from a 14-year-old male) were cultured in agarose gel
and treated for 3 days with interleukin-la (IL-la) and IL-I receptor antagonist protein (IRAP) at the concentrations shown. Synthesis of
3SS-proteoglycans(3sS-PGs),corrected for DNA content, was calculated as a percentage of that in untreated control cultures. Hatched bars
show superficial cells (from 10% of cartilage wet weight); open bars show the remaining deeper cells (from 90% of cartilage wet weight). Values
are the mean and SD (n = 3). Note that at its highest concentration, IRAP restored F G synthesis to the control value in cells from deep cartilage,
but failed to reverse the inhibitory effect of IL-1a in cells from the articular surface. These results are representative of 2 experiments on knee
cartilage from 2 different male donors (ages 14 and 55). B, Cartilage slices from the superficial and deeper layers of femoral condyles (from a
32-year-old and a 51-year-old male) were cultured with and without IL-la,IL-Ip, I M P , and with both an active cytokine and IRAP.
Concentrations of stromelysin and tissue inhibitor of metalloproteinases 1 (TIMP-I) in samples of media were measured by enzyme-linked
immunosorbent assay on the second day of treatment. Results shown for each treatment group are the TIMP-1:stromelysin ratio. Note that in
both deep and superficial cartilage, treatment with IL-la or p caused a profound lowering of the ratio, but this was more marked in the
superficial than in the deep cartilage. In deep cartilage treated with IL-laor B, the antagonist, I M P , was much more effective in restoring the
TIMP-1:stromelysin ratio toward control values than in tissue from the surface of the joint.
bition of PG synthesis could be overcome almost
completely with an 1RAP:IL-la molar ratio of 1,000
(Figure 3A). In contrast, in cultures of chondrocytes
from the superficial layer of cartilage, IRAP only
partially blocked IL-1*induced inhibition, with PG
synthesis being restored to -30% of that in control
cultures (Figure 3A). Similar results were obtained
using human articular chondrocytes from the superficial and deeper layers of knee cartilage (femoral
condyles) of a 55-year-old donor (data not shown).
Chondrocytes from the different layers were cultured
in agarose and treated under identical conditions as the
cultured chondrocytes shown in Figure 3A (femoral
condyles from a 14-year-old). In another experiment,
when the molar ratio of IRAPAL-1a was increased to
lO,OOO, the response of the superficial cells to IL-la
could be blocked almost completely (data not shown).
The concentrations of stromelysin, TIMP-1,
and PGE, in harvested culture media were measured
following treatment with IL-la and p; the results are
shown in Table 3. This preliminary examination of
conditioned medium showed that in the absence of
IL- 1 treatment, superficial cartilage slices secreted
significantly more PGE, than did deeper tissues (Table
3). From these results, we calculated the TIMP-1:
stromelysin ratios in culture media from superficial
and deep cartilage slices (obtained from 4 knees of 2
organ donors ages 32 and 51) that had been treated
with both active isoforms of IL-1; the results are
shown in Figure 3B. After treatment with IL-1a and p,
the TIMP-1:stromelysin ratio in the culture medium
decreased 26-fold in superficial slices and 9-fold in
deep slices, compared with control slices (Table 3).
Consistent with the results described above for PG
metabolism, IRAP was more effective in blocking IL-1induced responses in deeper cartilage than in superficial
cartilage. When cultures were treated simultaneously
with IL-laor pand IRAP,the TIMP-1:stromelysin ratio
reached control levels in cultures of deep cartilage, but
remained <50% of the control value for cartilage from
the surface of the joint (Figure 3B).
Receptors for IL-1. Specific binding experiments with 12sI-labeled IL-1a were performed on
chondrocytes isolated from the full thickness of femoral cartilage after 5 days of culture in alginate beads
(52). The results showed 1,OOO copies of the receptor
-
ARTICULAR SURFACE SUSCEPTIBILITY TO IL-I
483
Table 3. Effect of interleukin-la (IL-la) or IL-lp (0.5 ng/ml) and their inhibitor 1L-l receptor antagonist protein (IRAP) (500 ng/ml) on the
secretion of TIMP-1, stromelysin, and PGE, in media from cultured superficial and deep cartilage slices from human knee joints*
Superficial
TIMP-I
Stromelysin
SEZ
Deep
TIMP-I
Stromelysin
PGE,
+ IRAP
IL-Ip + IRAP
IL-1 a
IL- I p
IRAP
5.2
0.14
f 0.015
1.2 2 2.1
0.71 2 0.5
4.2 t 7.6
0.5 t 1.2
0.69 t 0.25
110.9 t 83.7
4.5 t 2.4
0.27 2 0.19
0.19 2 0.31
3 f 1.4
0.19 f 0.11
0.012 f 0.01
5.4 2 2.8
0.28 2 0.2
0.015 2 0.01
1.2
1.9 2 1.0
0.5 t 0.3
2.7 2 3.2
1.4 2 1.3
0.5 t 0.35
27.4 2 44.5
2.3 2 1.3
0.08 2 0.09
0.01 f 0.01
2.5 2 1.3
0.09 f 0.07
0.24 f 0.4
2.6 f 1.3
0.11 2 0.08
ND
Control
6.3
0.14
0.017
2
2
3.5
f
0.1 t 0.05
0.01 2 0.01
IL-la
* The concentrations in media harvested on the second day of treatment with cytokines are expressed as follows: for tissue inhibitor of
metalloproteinases 1 (TIMP-I) ng/ml; for stromelysin &d; and for prostaglandin E2(PGE,) pg/mY106 cells/24 hours. See also Figure 3B. ND
= not done. Values are the mean f SD (n = 4).
per cell, corresponding to an affinity of 0.9 X 10'oM-'
in a 1 binding-site model (Figure 4A). Similar results
with respect to the number of binding sites per cell and
the affinity of IL-1a to its receptor were obtained in a
second experiment on chondrocytes isolated from the
knee joints of a 29-year-old donor. At IL-la concentrations that inhibited PG synthesis by 50% (0.0030.015 ng/ml), fewer than 1% of the receptors were
occupied in these experiments for specific binding of
'251-labeled IL-1a. However, when the specific binding of 1251-labeledIL-1/3 was examined selectively on
1.oo
cells from the superficial and deep layers of human
articular cartilage obtained from the normal femoral
condyles of 2 male donors and 1 female donor, there
was a >Zfold difference in the number of maximal
binding sites of the low-to-intermediate affinity type
between these 2 cell populations (Table 4 and Figure
4B). Chondrocytes from the deep zone had only
- 1,800 copies of the intermediate-affinity type, while
chondrocytesfrom the superficial cartilage showed 4,760
? 2,280 (mean 2 SD) copies of the low-affinity type.
In contrast, we could not detect any significant
0
h
k
0.75
f
0.50
8
>
0
a,
v
D
50
0.25
m
i
0.0 0.5 1 0
B (molec./cell*)
0.00
0.0
'xl03
0.5
1.5
1.0
2.0
Free 1251-IL-la
(nM)
A
2.5
3.0
0.0
* x i 0-3
0.5
1.5
1.0
2.0
2.5
3.0
Free 1251-IL-1
p (nM)
B
Figure 4. A, Specific binding curve of 'zsI-labeled interleukin-I a (IL-la) for chondrocytes cultured in alginate, derived from the full-thickness
articular cartilage from the normal femoral condyles of a 42-year-old male. Inset, Scatchard plot derived from binding study based on 1
binding-site model. Ka = 0.92 x lO'OM-', and 960 2 52 siteskell (n = 3). Similar results were obtained in a second experiment using
chondrocytes derived from full-thickness articular cartilage of the normal femoral condyles of a 29-year-old male (K, = 10"M-' and 1,300
siteskell). B, Specific binding curve of 'z51-labeledIL-1p for superficial and deep chondrocytes cultured in alginate, derived from the normal
femoral condyles of a 17-year-old male. 0 = cells from the superficial layer (10% wet weight of cartilage); 0 = cells from the remaining, deeper
layers (WO
wet weight of cartilage). Inset, Scatchard plot. In both populations, binding data are compatible with binding sites of 2 affinities:
for superficialchondrocytes K, 1 = 1.25 t 0.04 x IOI'M-' with 1,OOO t 27 siteskell and K, 2 = 2.7 2 0.03 x 10"M-' with 3,500 t 42 siteskell;
for deep chondrocytes K, 1 = 1.11 2 0.1 x 10"M-' with 525 f 54 siteskell and K, 2 = 3 2 0.2 x IO'OM-' with 1,800 2 127 siteskell (n =
3). Similar results were obtained using superficial and deep chondrocytes derived from the normal femoral condyles of 2 other donors (ages 13
and 24) and cultured in alginate (see Table 4).
484
HAUSELMANN ET AL
Table 4. Specific binding sites and ailhities of IZSI-labeledIL-1s for superficial and deep chondrocytes derived from normal femoral condyles of 2 male donors and 1 female donor and cultured in alginate*
~
Superficial
Superficial
B,
Donor age
17
24
13
Mean f SD age,
18 f 5
Deep
Bmax 2
Bmax 1
1,cm
360
3,500
7,400
3,400
525
850
1,033
680
4,760 f 2,280t
803 f 257t
1
-
Deep
Ka 1
(XIO")
Ka 2
(XI04
Ka 1
(X10")
1,800
-
I .25
1.33
-
0.27
1.5
0.63
1.11
3.0
0.43
1,800
1.29
0.8 f 0.63t
B,
2
-
1.51
f
1.33t
Ka 2
( x 10'9
3.0
-
3.0
* Maximal binding sites (Bmx 1 and B,,, 2) and attinities (Ka1 and K, 2, in M-')of subsets of superficial and deep chondrocytes are shown.
Specific binding of "'I-labeled interIeukin-l@(IL-1s)
was determined in the presence of a 250-fold concentration of unlabeled IL-Ip. Binding
data from the chondrocytes of the 17-year-old donor are compatible with binding sites of 2 affinities in both populations of chondrocytes.
Binding data from the chondrocytes of the 24-year-old donor are compatible with 2 binding sites only in the superficial layer, whereas binding
data from the chondrocytes of both layers from the 13-year-old donor are compatible with binding sites of 1 affinity.
t Mean f SD.
difference between the number of the high-affinity
binding sites in these 2 cell populations (803 ? 257
binding sites in deep chondrocytes versus 680 in
superficial chondrocytes). Results of the Scatchard
plot indicated binding sites of lower and higher affinities in the superficial cells from 2 of 3 donors, and in
the deep chondrocytes from 1 of 3 donors. Importantly, the difference in affinity of IL-1p for the 2
classes of binding sites was almost 2 orders of magnitude (62-fold) in superficial cells, but only 1 order of
magnitude (20-fold)in deep cells (Table 4). In both cell
populations, the binding constant of the sites with high
affinity for IL-1p were almost 10 times higher than that
for sites binding IL-1a (Figures 4A and B and Table 4).
DISCUSSION
The results reported here demonstrate striking
metabolic differences in the responsiveness to IL- 1
between chondrocytes derived from superficial and
deep layers of normal human articular cartilage, and
show the value of analyzing separately these diverse
chondrocyte populations.
The specific results discussed below are from
experiments on normal human cartilage and chondrocytes from 18 donors, as described in Subjects and
Methods. Because of the fundamental difficulty of
obtaining sufficient samples of normal human articular
cartilage, it was not always possible to repeat each
experiment numerous times. This problem was compounded when the cartilage was subdivided into zones
to compare the responses of different populations of
chondrocytes, since yields of cartilage and cells were
lower. However, results of experiments with similar
samples were consistent, and supported the results
shown here. As indicated above, most experiments
were done 3 or 4 times, and a minimum of 2 times, with
similar results.
Cells and tissue slices from the articular surface
showed more severe suppression of PG synthesis in
response to IL-1 treatment than did cells and cartilage
from deeper layers. In comparison with chondrocytes
from the articular surface, at least a 10-fold greater
concentration of IL- 1(Y was required for similar inhibition of PG synthesis in cells from the deep layer.
While the superficial cartilage is more cellular than the
deeper tissue, our results for both cell and cartilage
slice cultures were corrected for content of DNA;
therefore, the difference in cellularity of the native
tissue cannot account for the observed variations in
responsiveness. The results also demonstrate that
human chondrocytes showed a consistent and significantly greater suppression of PG synthesis when
treated with IL-lp than with IL-la; this was the case
irrespective of the layer of the cartilage, as has been
recently reported elsewhere (58). These results were
supported by our findings that IL-Ip stimulated secretion of PGE, significantly moreso than did IL-la
(10-fold in chondrocytes from the deep zone and
25-fold in those from the superficial zone) (Table 3).
These results are also consistent with a recent report
that IL-la and p have different effects on the arachidonic acid cascade in cultured human articular chondrocytes (59), and they demonstrate a strong correlation between the effectiveness of the 2 isoforms of IL-1
to stimulate secretion of PGE, and inhibit PG synthesis in chondrocytes.
ARTICULAR SURFACE SUSCEPTIBILITY TO IL-1
Human articular chondrocytes from a 50-yearold donor responded to both isoforms of IL-1 with
only a weak catabolic response, and in this respect,
they differed significantly from the results of studies
utilizing mostly young bovine cartilage and chondrocytes (8,9). Other investigators have also observed a
weak catabolic response to 1L-1 in most samples of
human cartilage, but results vary with the donor, and
possibly also with the anatomical site (60) and age
(Bayliss MT: personal communication). The reason
for the common resistance of human cartilage to
showing a catabolic response to IL-1 is not understood, but the results suggest that apart from the
reasons discussed above, there may be different regulatory pathways for anabolic and catabolic events in
normal human articular chondrocytes. In addition,
other mediators may be required to permit the action
of IL-1 above the threshold response.
Ours were the first direct binding studies in a
3-dimensional culture system to show 2 classes of
binding sites with almost 2 orders of magnitude (62fold) difference in affinity for IL- 1p in human articular
chondrocytes of the superficial zone but only 1 order
of magnitude (20-fold) difference in deep zone cells.
This could be seen in 2 of 3 donors for superficial cells
and in 1 of 3 donors for deep cells. In contrast,
chondrocytes from at least 2 different donors appeared
to express only 1 class of binding sites of intermediate
affinity for IL-1a. This suggests that after stimulation
with IL-1p, both populations of human articular chondrocytes expressed 2 different classes of IL- 1 binding
sites, at least in 2 of 3 donors with respect to superficial chondrocytes and 1 of 3 donors with respect to
deeper chondrocytes. The reason for this variability is
not presently known.
In earlier studies on Raji B lymphoma and other
cells (29,30,33),there was a 10-100-fold stronger binding of IL-lp to type I1 IL-lR, in comparison with
1L-la and IRAP. For those experiments, equivalent
concentrations of recombinant IL-1a and p from different species, including human recombinant forms,
were used (31). In addition, our results revealed important differences in the number of receptors in
subpopulations of human chondrocytes. Chondrocytes from the surface of the cartilage had approximately twice the number of receptors for IL-1 with
respect to the low-affinity binding class in comparison
with cells from the deep cartilage of the same joint
(50). So far, published reports on IL-1 receptors of
chondrocytes cultured in monolayer have indicated a
single class of IL-1 receptor (61), but some differences
485
in affinity were reported. For example, rabbit chondrocytes, cultured in monolayers, contained approximately 1,620 receptors per cell, with high affinity for
IL-lp ( K , of 10'3M-'), corresponding to the 80-kd or
type I receptor (5435). While results of our binding
studies with IL-la and p (Figures 4A and B) support
these earlier findings of high-affinity IL-1 receptors on
articular chondrocytes (54), they also clearly show, for
the first time, that the IL-1 receptors present on human
articular chondrocytes from superficial and deep zones
possess different binding properties for IL-1p.
Whether only the type I receptor is present in chondrocytes or whether treatment with IL-1 causes an
upregulation of the type I1 receptor on certain cells
remains to be established. However, preliminary evidence from this laboratory points to the additional
presence of the 68-kd receptor (type I1 IL-1R) in
chondrocytes from both superficial and deep layers of
human articular cartilage following treatment with
IL-1 (Hauselmann et al: unpublished observations). It
has recently been reported that human synoviocytes,
cells which bear a close developmental and anatomical
relationship to articular chondrocytes, express both
type I and type I1 receptors for IL-1 (36).
Subpopulations of human chondrocytes from
superficial and deep layers of articular cartilage
showed significant differences not only in their responsiveness to IL-1 a and p, but also in their responsiveness to IL-1 in the presence of I M P . IL-14nduced
inhibition of PG synthesis was completely blocked by
IRAP in chondrocytes from the deep cartilage, but
only partially blocked in cells from the surface of the
cartilage. Chondrocytes from deep layers of cartilage
assemble much more extracellular matrix than do cells
from the cartilage surface. However, it is unlikely that
variations in the accumulation of matrix around the
chondrocytes could account for the observed differences in effective blocking of the IL-1 response by
IRAP; rather, the antagonist may have more ready
access to cartilage surface chondrocytes that have
sparse matrix. This difference in the effectiveness of
IRAP may instead result from a difference in binding
affinities of IRAP for the 2 isoforms of IL-1, together
with differences in the number and affinity of receptors
for IL-1 in the 2 cell populations.
In the joint, the synthesis of stromelysin and
TIMP are known to be influenced by the presence of
1L-1 (3,62,63). After treatment with IL-la and p, the
TIMP- 1:stromelysin concentration ratio in culture medium decreased 26-fold in superficial slices and 9-fold
in deep slices (compared with control slices), demon-
486
strating a significant increase in stromelysin and a
decrease in TIMP-1 concentration after treatment with
these cytokines. This change in the TIMP-1:
stromelysin ratio is very similar to the reported 20-fold
decrease of the TIMP-1:stromelysin ratio in synovial
fluid from patients with joint trauma compared with
synovial fluid from normal controls (64).In our cultures, the effectiveness of IRAP in blocking the IL-linduced decrease in the ratio of TIMP-1:stromelysin
varied according to the cell population and the active
isoform of IL-1. In this regard, IRAP was, again, less
effective as an antagonist in chondrocytes from the
articular surface than in those from deeper cartilage
(Table 3 and Figure 3B).
The data presented here emphasize the significant variations in the effectiveness of IRAP in blocking
specific IL-1 effects in these subpopulations of chondrocytes. Based on these results, we propose first, that
among the more abundant IL-1 receptors in chondrocytes at the surface of the articular cartilage, there is a
class which has a higher binding afhity to IL-lp and
can be less readily inhibited by IRAP. These cellular
differences in types and affinities of IL-1 binding sites
may, in part, explain the observed variations between
populations of chondrocytes. Second, the higher number of binding sites for IL-1p in the superficial layers,
compared with the deeper layers, of cartilage may
explain to some extent why IRAP can only partially
inhibit the effects of IL-la or p in most of our
experiments on mixed populations of human chondrocytes derived from full-thickness cartilage (data not
shown).
In our in vitro culture system, a 10-100-fold
molar excess of IRAP was needed to inhibit IL-1
responses to 50% of maximum. This finding is consistent with previous reports that blocking of IL-1induced inflammation or hypotension in vivo requires
as much as a thousand-fold molar excess of IRAP
(21,35). These results also support the claim that a
very small percentage of the IL-1 receptors (51%)
need to be occupied by IL-1 to trigger or sustain a
cellular response. By virtue of this highly effective
mechanism, IL-1 may indeed play a destructive role in
articular cartilage by downregulating anabolism during
attempted tissue repair. In addition, even if chondrocytes and synoviocytes bordering the joint cavity
produce IRAP itself, our results suggest that this
antagonist could give only partial protection against
the deleterious action of IL-1 on the articular surface.
HAUSELMANN ET AL
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