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Elevated substance p and accelerated cartilage degradation in rabbit knees injected with interleukin-1 and tumor necrosis factor.

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1023
ELEVATED SUBSTANCE P AND ACCELERATED
CARTILAGE DEGRADATION IN RABBIT KNEES
INJECTED WITH INTERLEUKIN-1 AND
TUMOR NECROSIS FACTOR
ELIZABETH M. O'BYRNE, VINCENT BLANCUZZI, DOUGLAS E. WILSON,
MARY WONG. and ARC0 Y. JENG
Cytokines, interleukin-1 (IL-I), tumor necrosis
factor a,and the neurotransmitter, substance P, have
been implicated in the pathogenesis of arthritis because
they stimulate synovial cells to secrete prostaglandin E,
and collagenase in vitro. We investigated in vivo changes
in intraarticular substance P and the degradation of
cartilage proteoglycan in response to intraarticular cytokine injections in rabbits. Twenty-four hours after a
single injection of 10 ng, 30 ng, or 100 ng of recombinant
human &la (rHuIL-la) per joint, the mean f SEM
levels of substance P detected in the cell-free joint lavage
fluid were 250 & 67 fmoles, 480 2 60 fmoles, and 530 &
130 fmoles (n = 4-9, respectively. The level of substance P in the contralateral knees injected with diluent
was 58 -c 8 fmoles (n = 12). The level of substance P had
increased by 2 hours after IL-1 injection and remained
elevated in the joint 48 hours after injection. Cytokineinduced proteoglycan depletion was also time- and dosedependent. Proteoglycan concentrations in articular
cartilage dissected from the weight-bearing condyles
were calculated as the ratio of sulfated glycosaminoglycan measured using 1,9-dimethyImethylene blue:
hydroxyproline. After 48 hours, 10 ng, 30 ng, or 100 ng
of rHuIL-la per joint decreased proteoglycan levels by
9 f 4%, 14 f 4%, and 21 2 3% (n = a), respectively. Likewise, the injection of recombinant human
-.
.____
From the Research Department. Pharmaceuticals Division,
Ciba-Geigy Corporation, Summit. New Jersey.
Elizabeth M.O'Byrne. PhD; Vincent Blancuzzi, MS; Douglas E. Wilson, BA; Mary Wong. BS; Arco Y. Jeng, PhD.
Address reprint requests to Elizabeth M. O'Byme. PhD.
InflammatiodOsteoarthritisResearch. Ciba-Geigy. 556 Morris Avenue. Summit NJ 07901.
Submitted for publication November I . 1989; accepted in
revised form February 20. 1990.
Arthritis and Rheumatism, Vol. 33, No. 7 (July 1990)
tumor necrosis factor a induced depletion of intraarticular substance P and cartilage proteoglycan.
Rheumatoid arthritis (RA) and osteoarthritis
(OA) are both multifactorial diseases which lead to
joint degeneration. Arthritis is characterized by inflammation and erosion of the articular cartilage. The
cartilage proteoglycan matrix gives cartilage resilience
and elasticity, allowing distribution of mechanical
forces in the weight-bearing joint. The loss of the
cartilage proteoglycan matrix is an early event in the
joint destruction seen in RA and OA.
Fell and Jubb ( I ) first noted that the synovium
elicits mediators that stimulate cartilage chondrocytes
to degrade their surrounding extracellular proteoglycan. One of these mediators has been identified as
interleukin-la (1L-la) (2). IL-I increases cartilage
chondrocyte production of cartilage-degrading enzymes and prostaglandin E, (PGEJ and depresses
proteoglycan biosynthesis (3.4).
Cartilage degradation induced by tumor necrosis factor (TNF) has also been observed ( 5 ) . The
IL-l-stimulated neutral protease release from synoviocytes and articular chondrocytes and the promotion of bone resorption may initiate the progressive
degenerative changes in OA (6). In addition to these
effects on the joint, IL-I also modulates immune
function and mediates inflammation by augmenting T
cell proliferation to antigens and by inducing PGE,
production (7); it also stimulates the central nervous
system to induce fever (8).
A role of the nervous system in inflammation
and joint degeneration has also been suggested. A
majority of RA patients are diagnosed as having symmetric joint swelling (9). Patients who develop RA
1024
O'BYRNE ET AL
after sustaining paralyzing lesions t o the nervous system are spared joint inflammation in the paralyzed
limbs (10). Furthermore, the relative density of innervation may account for the more frequent and more
severe involvement of specific joints in RA (10).
Macrophages ( l l ) , neutrophils (12), and T and B
lymphocytes (13) that are engaged in inflammatory
responses have been identified as targets of the neuropeptide, substance P. Substance P may modulate
local immunologic and inflammatory responses in the
arthritic joint when released by peripheral nerves in
the synovium in response t o inflammatory stimuli (14).
Endogenous levels of 1L-l may be partially
regulated by substance P. Substance P has been reported to stimulate synovial production of PGEz and
collagenase (151, and monocyte secretion of IL-I and
TNF (16). Substance P also induces secretion of IL-1
activity by P388D1 cells (17). These studies of cells
suggest that, by inducing IL-1 and TNF, substance P
may play a role in the tissue damage in chronic
inflammation and OA.
In animal studies, the injection of IL-1 can
induce inflammation and cartilage proteoglycan loss
(18-23), as well a s cause fever (24) in rabbits. Whether
or not substance P is produced in vivo after IL-1
injection has not been reported. In the present study,
intraarticular levels of substance P were measured
after injection of recombinant human I L - l a (rHulLl a ) and r H u T N F a in rabbit knees. Substance P levels
were elevated, with a concomitant loss of cartilage
proteogl ycan.
MATERIALS AND METHODS
Materials. New Zealand white rabbits (2.5-3.0 kg)
were purchased from HARE, Inc. (Hewitt. NJ). Substance P
and substance P antisera were from Cambridge Research
Biochemicals (Valley Stream, NY). Goat anti-rabbit immunoglobulin antibodies and normal rabbit serum were purchased from Calbiochem (La Jolla. CAI. '251-labeled substance P was obtained from New England Nuclear (Boston.
MA). Polyethylene glycol (PEG) 8000 was purchased from
Sigma (St. Louis. MO). Hyaluronidase was obtained from
Cooper Biomedical (Malvern, PA). The rHuIL-la and
rHuTNFa were supplied by Biogen (Geneva, Switzerland)
and AmGen Biologicals (Thousand Oaks, CA). respectively.
Cytokines were stored at -70°C in 20 &ml of 1% human
serum albumin in phosphate buffered saline (PBS) until use.
In designated experiments, rHuIL-la was denatured by
boiling for 30 minutes.
Intraarticular iqjections. For the injection of cytokines. in 0.1 ml of PBS containing 0.1% heat-inactivated
normal rabbit serum, a 26gauge half-inch needle was inserted through the suprapatellar ligament into the joint space
(22). The contralateral knee joint was injected with an equal
volume of the vehicle.
Joint lavage. To lavage the joint, I ml of isotonic
saline was injected into the joint space through the suprapatellar ligament, the knee was flexed 10 times, and the lavage
fluid was withdrawn with a syringe and a 20-gauge 1" needle.
Typically, 700 pl of the lavage fluid was recovered. An
aliquot of 40 pl ofjoint lavage fluid was mixed with 20 ml of
isoton and 5-6 drops of Zap-0-globin, and the cell numbers
were counted in a Model ZBI Coulter Counter (Coulter,
Hialeah. FL). Cytocentrifuge slides were prepared by centrifuging a 200-pI aliquot of joint lavage fluid diluted to 1 x
106 ceIIs/d at 700 revolutions per minute for 7 minutes in a
Cytospin centrifuge (Shandon Southern Products, Puncorn,
Cheshire. UK). Slides were stained in an HMS 360 stainer
(Honeywell, Denver, CO) using azure Blmethylene blue/
eosin (Hemal Stain Co.. Danbury. CT). The remaining joint
lavage fluid was centrifuged in a Hermle Z 365 centrifuge
(Berthold Hermle GmbH and Co., Gosheim. FRG) at 12,000
rpm (13.6OOg) for 10 minutes to pellet the cells. This cell-free
joint wash was analyzed for substance P.
Measurement of substance P by radioimmunoassay
(RIA). Vials of substance P antiserum. goat anti-rabbit
immunoglobulin antibodies, and normal rabbit serum obtained commercially were reconstituted with an RIA buffer
containing 0.2% bovine serum albumin, 0.1% Triton X-100.
and 0.02% NaN, in PBS. The substance P RIA was carried
out at room temperature as described elsewhere (25). with
modifications. The viscosity of the joint lavage fluid due to
hyaluronic acid (HA) prevented samples from pelleting in
the substance P RIA. using a PEG preciditation method.
Thus, a prerequisite for measuring substance P in the joint
fluid by the use of an RIA is to reduce the sample viscosity
by digesting the HA in the synovial fluid with hyaluronidase.
The effect of hyaluronidase on the substance P assay was
examined. Treatment of cell-free lavage fluid samples (100
pl) from control knees with up to 1 unit of hyaluronidase for
I hour at 37°C did not interfere with the results of the
substance P assay. Higher concentrations of hyaluronidase
caused a decrease in the amount of substance P that bound
to the antibodies. When the cell-free joint lavage was treated
with 100 units of hyaluronidase, only 40% of the substance P
was present. as compared with the untreated control. It is
possible that the commercial hyaluronidase contains peptidases that degrade substance P.
Several commercially available hyaluronidases were
also tested. Hyaluronidase from Cooper Biomedical appeared to be satisfactory in reducing the viscosity of the
lavage samples, as well as giving minimum interference in
the substance P assay. The standard curve for substance P in
the RIA using nonradioactive substance P treated with 0.2
units of hyaluronidase gave identical results as compared
with untreated controls.
For routine analysis. 100-plaliquots of cell-free joint
lavage fluid samples were treated with 10 p1 of hyaluronidase
(0.2 units) at 37°C for I hour to digest the HA. These samples
were then mixed with 200 pI of RIA buffer, 100 pI of
substance P antiserum (diluted 1:200), and 100 pl of
labeled substance P (10,OOO counts per minuteltube). After 2
hours of incubation, 100 p1 of goat anti-rabbit immunoglobulin antibodies and 100 pI of normal rabbit serum were added
-
EFFECTS OF IL-1 A N D TNF ON SUBSTANCE P
and incubated further for 45 minutes. The antigen-antibody
complex was precipitated with 1 ml of 17.5% PEG and
centrifuged in a tabletop centrifuge. The supernatant was
decanted, and the radioactivity in the pellet was counted in
a gamma counter. Total binding and nonspecific binding
were measured in the absence of nonradioactive substance P
and substance P antiserum, respectively; in general, these
values were 6,000 cpm and 300 cpm. respectively.
Cartilage proteoglycan concentration. The articular
cartilage was dissected from the weight-bearing surfaces of
the femur and digested with papain (26). Sulfated glycosaminoglycan (GAG-SO,) was measured using the 1.9dimethylmethylene blue assay (26). Hydroxyproline content
was quantitated in the cartilage digest (27.28). Changes in the
percentage of cartilage proteoglycan in the cytokine-injected
knees versus the vehicle-injected knees were calculated
from the GAG-S0,:hydroxyproline ratio, using each rabbit
as its own control (29).
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RESULTS
Synovial joints are lubricated with a fluid rich in
HA. Therefore, joint lavage samples were routinely
incubated with 0.2 units of hyaluronidase for 1 hour at
37°C. as described in Materials and Methods. The
substance P RIA detects only biologically active substance P peptides. The biologically inactive substance
P peptide 7-11 fragment (30) was 200-fold weaker in
the substance P RIA than was the native substance P
or the biologically active 4-1 1 fragment.
The substance P levels in joint fluid collected 24
hours after a single injection of vehicle o r of 10 ng. 30
ng, or 100 ng of r H u I L - l a indicate a dose-dependent
increase in intraanicular substance P (Figure la). In
more than 90% of the vehicle-injected knees, the
substance P level was below the limits of detection by
the RIA (40 fmoles/joint). The displacement of radioactive substance P was <lo% in the rest of the
samples from the vehicle-injected knees in which
substance P was detectable. After injection of 10 ng,
30 ng, or 100 ng of r H u I L - l a per knee, mean 2 SEM
substance P levels of 250 k 67 fmoles, 480 I60
fmoles, and 530 k 130 fmoles per joint, respectively,
were detected. An increase in cellular inflammation
was also observed (Figure Ib). Interleukin-1-induced
cartilage proteoglycan depletion was dose- and timedependent. At 24 hours, up to 30 ng of rHuIL-la did
not significantly decrease the proteoglycan content,
whereas 100 ng of r H u I L - l a per knee decreased the
proteoglycan concentration by a mean 5 SEM of 35 I
9%. After 48 hours, 10 ng, 30 ng. and 100 ng of
rHulL- 1a per joint decreased proteoglycan concentration in femoral cartilage by 9 2 4%. 14 5 4%. and 21
+- 3% (n = 8). respectively (Figure Ic).
26
T
Ong
long
30ng
100ng
Figure 1. Biologic responses to single intraarticular injections of
recombinant human interleukin-l a (rHulL-1 a) in rabbit knees.
Substance P (a) and cell counts (b) were measured 24 hours after
intraarticular injection of various amounts of rHulL-la as indicated.
Cartilage proteoglycan loss (c) was measured 48 hours after injection. Values are the mean 2 SEM for 5 rabbits in each treatment
group. The control group consisted of the 15 vehicle-injected
contralateral knees.
O’BYRNE ET AL
1026
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Substance P is released into the joint soon after
IL-I injection. Two hours after the injection of 100 ng
of rHulL-1 a, a significant increase in intraarticular
substance P was measured (Figure 2). At 6 hours, the
substance P level in the joint reached the same level as
observed at 24 hours (Figures la and 2). Polymorphonuclear neutrophils were the predominant cell type in
the joint fluid 2-24 hours after IL-I injection
(21.23,28). At 24 hours, some mononuclear cells appeared in the joint; at 72 hours, mononuclear cells
accounted for the majority of the joint inflammation
(Table 1).
Intraarticular substance P remained elevated 48
hours after a single injection of 100 ng of rHuIL- I a or
100 ng of TNFa (Figure 3). Injection of TNFa has also
been shown to cause cellular infiltration and cartilage
proteoglycan loss, comparable with that seen in reTable 1. Distribution of cells in rabbit knee joint lavage fluid at
different times following intraarticular injection of 100 ng of recombinant human interleukin-1a
4
24
48
72
n
8
5
8
I
8
Total cells
( X 10- 6 ) .
mean ? SEM
0.6 5
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25.3 2
6.5 +.
2.9 2
%
neutrophils
0.1
3.2
4.4
2
96
69
1.3
1.0
41
38
-
0
6hr
Figure 2. Initial time course of changes in levels of substance P in
the joint after intraarticular injection of recombinant human interleukin-la (rHulL-la). Substance P in joint lavage fluid was measured by radioimmunoassay 2 hours, 4 hours, and 6 hours after
injection of 100 ng of r H u l L - l a per knee. Values are the mean 5
SEM of 4 rabbits for each time point.
0
200-
v)
100
Hours after
interleukin-1
injection
400-
%
monocytes
98
4
31
62
59
lOOng IL-1
l O O n g TNF
4 8 HOURS POST INJECTION
Figure 3. Levels of intraarticular substance P in rabbit knees injected with recombinant human interleukin- la (rHulL-1a)or recombinant human tumor necrosis factor a (rHuTNFa). Substance P in
joint lavage fluid was measured by radioimrnunoassay 48 hours after
injection of 100 ng of rHuIL-la or rHuTNFa. Values are the mean
? SEM of 8 rabbits per treatment group.
sponse to 1L-1 (29). The decrease in cartilage proteoglycan concentration is transient and is followed by
regeneration within a week of IL-I injection (18-23).
Heat-denatured rHulL-la at 100 ng/ml of cul-
l
1200
1
1000
1100
.-C
0
sooj
7
g
800
K
700
0
E
800
i
T
IL-1
H* IL-1
Figure 4. Levels of intraarticular substance P induced by biologically active and heat-inactivated ( H I ) recombinant human interleukin-la (rHulL-a). Substance P was measured by radioimmunoassay
24 hours after 3 daily injections of 100 ng of native or heat-denatured
rHulL-la. Solid bars represent controls (vehicle injected); hatched
bars represent groups treated with active or inactivated rHulL-la.
Values are the mean ? SEM of 8 rabbits per treatment group.
EFFECTS OF IL-1 AND TNF ON SUBSTANCE P
ture medium was inactive, while native rHuIL-la at 1
ng/ml of culture medium stimulated a 3-fold increase in
proteoglycan release in an organ culture cartilage
degradation assay (4). Only biologically active rHuILla induced an increase in intraarticular substance P.
Injection of heat-inactivated rHuIL- 1 a did not cause
an increase in the level of intraarticular substance P
(Figure 4) or a decrease in the cartilage proteoglycan
concentration (results not shown).
DISCUSSION
Our data clearly demonstrate that intraarticular
administration of the cytokines, rHuIL-l a or
rHuTNFa, led to an increased level of substance P in
the joint. Levels of biologically active substance P in
the joint remained elevated for at least 48 hours after
injection. Therefore. in addition to the induction of
IL-I by substance P (l6,17), IL-1 also triggered substance P release in the inflamed joint.
In separate experiments, substance P was not
detected in rabbit knees 24 hours after intraarticular
injection of 1 pg of substance P. These experiments
indicate that a bolus injection of substance P does not
create sustained exposure of synovial tissues to substance P. However, a single injection of rHulL-l or
rHuTNFa resulted in prolonged exposure of joint
tissues to substance P.
The chain of events that led to substance P
release following cytokine injection may be a direct
action of cytokines on afferent sensory neurons, or a
secondary response to inflammatory mediators such as
PGE, or invading leukocytes. This increased level of
substance P after injection of 1L-1 andor TNF, coupled with recent reports that substance P stimulates
production of 1L-I and TNF (15-17), may represent a
positive feedback between cytokines and substance P
in joint inflammation and cartilage degeneration. The
role of substance P in cytokine-induced inflammation and cartilage degradation and the mechanism of
cytokine-induced substance P release will be investigated in future experiments.
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