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The effect of an interleukin-1 receptor antagonist protein on type ii collageninduced arthritis and antigen-induced arthritis in mice.

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ARTHRITIS & RHEUMATISM Volume 36
Number 9, September 1993, pp 1305-1314
0 1993, American College of Rheumatology
1305
THE EFFECT OF AN
INTERLEUKIN-1 RECEPTOR ANTAGONIST PROTEIN
ON TYPE I1 COLLAGEN-INDUCED ARTHRITIS AND
ANTIGEN-INDUCED ARTHRITIS IN MICE
PAUL H. WOOLEY, JANEY D. WHALEN, DARRYL L. CHAPMAN, ANN E. BERGER,
KAREN A. RICHARD, DANIELLE G. ASPAR, and NIGEL D. STAITE
Objective. To investigate the anti-arthritic effect
of recombinant human interleukin-1 receptor antagonist
protein (IRAP) in two experimental models of arthritis.
Methods. Recombinant IRAP was administered
daily to mice with type I1 collagen-induced arthritis
(CIA) or with antigen-induced arthritis (AIA) provoked
by methylated bovine serum albumin (mBSA). Disease
incidence and severity were assessed by a clinical index
and histologic features. Serum antibody to type I1
collagen, spleen cell proliferation to mBSA, and antiIRAP antibodies were measured as indices of immune
function.
Results. IRAP reduced the incidence and delayed
the onset of CIA and suppressed the antibody response
to type I1 collagen. In contrast, IRAP did not affect the
pathogenesis of AIA and had no effect on either humoral
or cellular immune responses to mBSA in arthritic mice.
Conclusion. These observations suggest that
interleukin-1 may play a prominent role in the development of some, but not all, forms of arthritis.
Interleukin-1 (IL-1) is present in the synovial
fluid of rheumatoid arthritis (RA) patients (1-3). In
approximately 40% of joint fluids from patients with
RA, osteoarthritis, and miscellaneous connective tisFrom the Department of Internal Medicine and Immunology, Wayne State University Medical School, Detroit, and the
Departments of Cell Biology and Hypersensitivity Diseases Research, Upjohn Laboratories, Kalamazoo, Michigan.
Paul H. Wooley, PhD; Janey D. Whalen, BS; Darryl L.
Chapman, BA; Ann E. Berger, PhD; Karen A. Richard, MS;
Danielle G. Aspar, MA; Nigel D. Staite, PhD.
Address reprint requests to Nigel D. Staite, PhD, Department of Hypersensitivity Diseases Research, Upjohn Laboratories,
301 Henrietta Street, Kalamazoo, MI 49001.
Submitted for publication December 4, 1992; accepted in
revised form March 16, 1993.
sue diseases, IL-1 levels are significant (4). In situ
hybridization studies have demonstrated that IL-l p is
present in the rheumatoid synovium (5). In addition,
mononuclear cells derived from rheumatoid pannus
spontaneously produce IL-la and IL-1p (6), and IL-1
levels in the serum of RA patients correlate to some
extent with disease severity (7). These findings, together with the broad spectrum of biological activity of
IL-1 on chondrocytes, synovial fibroblasts, lymphocytes, and monocyte/macrophages, suggest that IL-1
could play a key role in the pathogenesis of rheumatic
disease.
Studies examining the arthritogenic activity of
IL-1 in animals have shown that direct administration
of IL-1 into normal rabbit or mouse knee joints causes
a mild and transient inflammatory reaction, accompanied by a temporary loss of cartilage proteoglycan
(8-10). In experimental models of arthritis, systemic
administration of IL- 1 accelerates and exacerbates
collagen-induced arthritis (CIA) in mice (1 1,12) and
streptococcal cell wall-induced arthritis in rats (13)
and will transform a mild inflammatory reaction induced with methylated bovine serum albumin (mBSA)
into an acute inflammatory and erosive arthritis (14).
These studies have focused on the effects of exogenous IL-1, however; the role of endogenous IL-1 in the
pathogenesis of experimentally induced arthritis has
not been widely studied (15,16).
IL-1 activity in vivo may be regulated by an
IL-1 receptor antagonist protein (IL-1rdIRAP) which
has been cloned from human monocytes and U937
(17-19). This protein inhibits the binding of IL-1 to its
receptor on T cells and inhibits IL-1-induced prostaglandin E, production by fibroblasts (17-20). In the
present study, we investigated the effects of IRAP on
WOOLEY ET AL
1306
two models of murine arthritis. Our results show that
IRAP has different effects on collagen-induced and
mBSA-induced arthritis. This suggestion of a differential role of IL-I in these two models could have
implications for the treatment of human RA with IL-I
antagonists.
MATERIALS AND METHODS
Animals. Female DBN1 LacJ mice (7-8 weeks old,
weighing 18-23 gm) were obtained from Jackson Laboratories (Bar Harbor, ME) for use in the CIA studies. Animals
were housed in the Department of Laboratory Animal Resources, Wayne State University, in accordance with the
animal care guidelines of the National Institutes of Health.
Mice were identified by ear notch and were quarantined for
2 weeks before the study began. For the antigen-induced
arthritis (AIA) studies, 8-10-week-old female C57BY6 mice
(Harlan-Sprague-Dawley, Indianapolis, IN) were used.
These animals were housed at Upjohn. Animals were serologically negative for mouse hepatitis, Sendai, and Minute
viruses throughout the study.
Induction of collagen arthritis. Chick type I1 collagen
(Genzyme, Boston, MA), prepared according to the method
of Cremer et a1 (21), was solubilized at 2 m g h l in 0.01M
acetic acid at 4°C overnight. Collagen was emulsified in an
equal volume of cold Freund’s complete adjuvant (Difco,
Detroit, MI) supplemented with 2 mg/ml of heat-killed Mycobacterium tuberculosis, strains C, DT, and PN mixed
(Central Veterinary Laboratory, Weybridge, UK). The
DBN1 LacJ mice were immunized intradermally at the base
of the tail with 100 pl of emulsion.
Induction of antigen arthritis. A monarticular arthritis
was induced in C57B116 mice with mBSA as previously
described (22,23). Briefly, mice were immunized (on day
-21) with 100 pl of 1 mg/ml mBSA (Sigma, St. Louis, MO)
emulsified in Freund’s complete adjuvant supplemented with
1 mglml heat-killed M tuberculosis, strains C, DT, and PN
mixed. Mice were reimmunized after 7 days. After a further
14 days (on day 0), mice were anesthetized with methoxyflurane (Metofane; Pitman-Moore, Washington Crossing,
NJ), and the skin over the left rear knee was cut to expose
the stifle joint. Next, 200 pg of mBSA in 20 pl of sterile
saline was injected into the knee joint, and the wound was
closed with a 4-mm wound clip. Animals were killed on day
14, and the injected knee was removed for histologic evaluation of the arthritis.
Administration of IL-1 antagonist protein. Saline vehicle, containing 0.5% autologous DBAh or C57B1/6 mouse
serum in 0.9% saline (pH 7.2) was sterilized before use by
passage through a 0.22-pm filter (Millipore, Bedford, MA).
Recombinant IRAP (rIRAP-U 1;batch 12/89)was cloned and
expressed from the monocytic cell line U937, at Upjohn
Laboratories (19). The protein was 99.9% pure by
Coomassie brilliant blue staining of sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis gels and had a
molecular weight of 17,143 daltons. The level of endotoxin
contamination was 0.12 n g h g of protein, as measured by a
chromogenic Limulus amebocyte lysate assay (Whittaker
Bioproducts, Walkersville, MD). In an IL- I/3 receptor binding assay using human YT cells, the Ki(inhibition affinity
constant) of the protein was 126 k 18 pM (mean ? SD). The
bioactivity of the protein, as measured by inhibition of IL-2
production from the murine T cell line LBRM 33.1A5, was
25,000-50,000 inhibitory unitshg. One inhibitory unit was
defined as the amount of IRAP required to cause 50%
inhibition of the response to 1 lymphocyte-activating factor
unit (LAF unit) of recombinant human IL-lp.
IRAP or human serum albumin (HSA; Sigma) was
diluted in saline vehicle at 3 mg/ml and stored in aliquots at
-80°C prior to use. Prednisolone (Sigma) solution was
prepared daily by dissolving dry crystals in a minimal
volume of ethanol followed by dilution to 0.75 mglml in
saline vehicle. DBN1 LacJ mice were injected intraperitoneally with 100 pl of test substance, once a day from day 14
after collagen immunization until the end of the study.
C57BY6 mice were dosed daily using 1 of 3 protocols: day
-21 to day 0, day -7 to day 14, and day 0 to day 14.
Assessment of arthritis. Mice with CIA were examined daily for the presence of arthritis in peripheral joints.
Disease was evaluated using a severity index previously
described (24), and each limb was scored 0 (normal appearance and flexion), 1 (erythema and edema), 2 (visible joint
distortion), or 3 (ankylosis detected on flexion), according to
the clinical assessment. Paw widths were measured using
constant-tension calipers. Animals were also weighed regularly throughout the study.
Joint pathology in mice with mBSA-induced arthritis
was assessed by histologic examination of the affected knee
joint. The extent of inflammation, pannus formation, cartilage damage, and bone erosion were graded separately, each
using a scale of 1.0 (mild) to 5.0 (severe). The inflammation
score was derived from evaluation of soft tissue inflammation, synovitis, cellular infiltration, and angiogenesis. The
extent of pannus formation was defined as hypertrophic
synovial tissue forming a tight junction with the articular
surface proximal to the margin of the epiphyseal cartilage
growth plate. Cartilage disruption, loss of matrix, and destruction was scored on both condylar surfaces, to yield the
cartilage damage score. The area and depth of erosion of the
subchondral bone were evaluated for the bone erosion score.
Scores were combined to give a global arthritis score (maximum 20) for each mouse, and these scores were averaged
for each group of 10 mice.
Measurement of serum anticollagen antibodies. Blood
was obtained by cardiac puncture of anesthetized animals at
the termination of the study. Blood samples were allowed to
clot, and the sera were removed and stored at -80°C until
assayed. A modified enzyme-linked immunosorbent assay
(ELISA) technique was used to detect anticollagen antibodies (25). ELISA plates (Nunc Immunoplate 11; Hazleton
Research Products, Denver, PA) were coated with 100 pl of
30 p g h l of bovine type I1 collagen dissolved in 0.4M
phosphate buffered saline (PBS), pH 7.6, for 18 hours at 4°C.
Plates were washed 3 times with PBS containing 0.05%
Tween 20 (PBS-Tween) and were blocked with 1% bovine
serum albumin (Sigma) for 1 hour at 4°C. Serum samples
were diluted 1:200 in PBS-Tween-BSA, and 100 pl was
added to the plates for 18 hours at room temperature. After
washing with PBS-Tween, 100 pl of alkaline phosphatase-
IL-1 ANTAGONIST PROTEIN IN MURINE ARTHRITIS
conjugated goat anti-mouse immunoglobulin (Fisher, Springfield, NJ) was added for 1 hour at 37°C. The plates were then
washed 10 times with PBS-Tween, and p-nitrophenyl phosphate substrate (Sigma), dissolved in diethanolamine buffer,
was added for 20 minutes at room temperature.
The reaction was stopped with 100 pl of 3N NaOH,
and the absorbance of each well was read at 405 nm using a
UV-max spectrophotometer (Molecular Devices, Menlo
Park, CA). Standard positive and normal (negative) mouse
sera were included as controls to check the reproducibility
and specificity of the assay.
Measurement of serum anti-mBSA antibodies. Nunc
96-well microtiter plates were coated for 24 hours at room
temperature with 100 pg/ml mBSA dissolved in water. After
3 washes with PBS, pH 7.2, containing 0.002% Tween 20,
100 pl of serum, diluted in PBS, was added to duplicate wells
for 2 hours at room temperature. For determination of IgM,
the incubation was for 24 hours at 4°C.
Assay plates were washed 3 times with PBS-Tween,
except for IgM plates, which were washed once with PBSTween containing 5% non-fat milk, followed by 2 washes in
PBS-Tween. Alkaline phosphatase-conjugated sheep antimouse IgGl, IgG2a, IgG2b, or IgM, or goat anti-mouse IgG3
(100 pl; Serotec, Oxford, UK) diluted in PBS-Tween was
then added. After 2 hours at room temperature, the plates
were washed twice with PBS-Tween and once with distilled
water. Alkaline phosphatase substrate (100 pl; Sigma) dissolved in diethanolamine buffer, pH 9.8, was added for 30
minutes at room temperature, and the color reaction was
halted with 100 pl of 3N NaOH. The absorbance of each well
was read at 405 nm against control wells using a Titertek
ELISA plate reader (Flow Laboratories, McLean, VA).
Spleen cell prolieration assay. Spleens were removed
from mBSA-immunized arthritic and normal (nonimmunized) C57BV6 mice 14 days after the induction of arthritis.
Spleens were gently teased apart, the erythrocytes lysed
with Tris ammonium chloride buffer, and single-cell suspensions prepared in Click’s EHAA medium supplemented with
0.5% C57BV6 serum, 2 mM glutamine, and 1,000 units of
penicillin, 1 mg/ml streptomycin (Gibco, Grand Island, NY),
and 5 x lO-’M 2mercaptoethanol. Cells were cultured in
96-well microtiter plates at 5 x lo5 cells/well in 200-pl
volumes and were stimulated with mBSA at various concentrations. Plates were incubated for 4 days at 37°C in a
humidified atmosphere containing 5% CO,. Each well was
pulsed with 0.5 pCi of tritiated thymidine (specific activity
2.0 Ci/mmole; Amersham, Amersham, UK) for the last 18
hours of culture. Cell proliferation was measured using the
Beta Plate liquid scintillation spectroscopy system (LKBWallac, Turku, Finland).
Measurement of serum anti-IRAP antibodies. AntiI M P antibodies were measured by ELISA. Costar EIA
half-area well plates were coated with 50 pl of IRAP at 2
p g h l in 0.015M bicarbonate buffer, pH 9.6, for 16 hours at
4°C. Wells were washed 3 times with PBS-Tween and were
blocked with 100 pl of PBS-Tween containing 1% gelatin for
1 hour at 18°C. After 3 washes with PBS-Tween, 50 pl of test
serum diluted in PBS was added for 1 hour at 18°C. After 3
further washes with PBS-Tween, 50 pl of a 1:1,OOO dilution
of goat anti-mouse IgG conjugated to horseradish peroxidase
(Kirkegaard & Perry, Gaithersburg, MD) was added for 1
1307
Table 1. Incidence of type I1 collagen-induced arthritis in IRAPtreated mice*
Treatment
group
Control
Saline vehicle
HSA (300 pg/day)
IRAP
3 &day
30 &day
300 &day
Prednisolone (75 pg/day)
Incidence of arthritis
Expt. 1
Expt. 2
8/10
6/10
5/10
9/10
7/10
8/10
3/10
1/10$
0/10$
0/10$
mot
NT
* The incidence of arthritis in DBNl LacJ mice was assessed 48
days (expt. 1) and 93 days (expt. 2) after immunization with chick
type I1 collagen. Mice were dosed daily with interleukin-1 receptor
antagonist protein (IRAP),by intraperitoneal injection, beginning on
day 14 after immunization, and continuing throughout the study. NT
= not tested.
t P < 0.02 versus saline and human serum albumin (HSA) controls,
by chi-square contingency analysis.
$ P < 0.005 versus saline and HSA controls, by chi-square contingency analysis.
hour at 18°C. Following 3 washes with PBS-Tween, 50 pl of
ABTS substrate was added. After 20 minutes, the reaction
was stopped with 50 pl of 1% SDS, and the optical density
was read at 405 nm.
A positive control serum was obtained from
(BALB/cJ x A/J)F, mice immunized initially with 7 pg of
IRAP emulsified in Freund’s complete adjuvant, and reimmunized twice with IRAP in Freund’s incomplete adjuvant.
These mice were then challenged with an intraperitoneal
injection of 5 pg of IRAP in saline, and serum was collected
3 days later.
Statistical analysis. Differences between the incidence of collagen arthritis in experimental groups were
tested for significance by a chi-square 2 x 2 contingency
analysis. Differences between time of onset of disease,
disease severity index, peak paw width, and anticollagen
antibody data were tested for significance using a one-way
analysis of variance (ANOVA). Histologic scores in mBSAimmunized arthritic mice were also analyzed by one-way
ANOVA.
RESULTS
Effect of IRAP treatment on the incidence of
CIA. Groups of 10 mice were given daily intraperitoneal injections of either saline vehicle, HSA, IRAP, or
prednisolone, in 2 separate experiments. Mice were
dosed from day 14 after collagen immunization until
the termination of the studies, on day 48 and day 93,
respectively.
As shown in Table 1, there was no significant
difference between the incidence of arthritis in the
saline vehicle-treated and HSA-treated groups in ei-
WOOLEY ET AL
1308
ther experiment. In the first study, IRAP given at 3 and
30 pg/day had little effect on the incidence of arthritis,
which was not significantly different from that in either
the saline or HSA control groups. In the second study,
a reduced incidence of arthritis was seen with both 3
and 30 pg/day IRAP, although a statistically significant
effect was only seen at the 30 pglday dosage. Notably,
only 1 mouse developed arthritis in the 300 pg/day
IRAP treatment group, and in the repeat study IRAP
completely inhibited the development of arthritis.
These reductions were highly significant compared
with the saline and HSA control groups (P < 0.005).
Treatment with 75 pglday of prednisolone completely
inhibited the development of arthritis and served as a
positive treatment control.
Effect of IRAP treatment on the onset of CIA.
Although treatment with 3 and 30 pg/day of IRAP
failed to alter the final incidence of CIA in the first
study, the onset of paw swelling was significantly
delayed in some groups. The mean day of onset for
saline and HSA control groups was approximately the
same. Treatment with 3 and 30 pg of IRAP daily
resulted in a significant delay in the onset of arthritis
compared with the saline vehicle group (Table 2). Only
the 30 pg IRAP treatment group was significantly
different from the HSA controls. The 1 affected mouse
in the 300 pg group developed arthritis on day 37, at
approximately the same time as the mice in the control
groups. Arthritis was first detected on days 19 and 23
Table 2. Mean day of onset of collagen-induced arthritis in IRAPtreated mice*
Treatment
group
Control
Saline vehicle
HSA (300 pglday)
IRAP
3 PgldaY
30 I d d a y
300 PimY
Prednisolone (75 &day)
Day of onset,
mean f SD
Expt. 1
Expt. 2
32.4 i 5.2 (8)
36.0 k 5.3 (9)
55.5
f 7.6 (6)
51.8
f 9.2 ( 5 )
39.4 2 5.7t (7)
41.4 2 2.5.t (8)
37.0 2 0 (1)
61.0 f 6.9 (3)
65.0 t 0 (1)
No arthritis
No arthritis
NT
* Groups of 10 DBNl LacJ mice were immunized with type I1
collagen on day 0. Mice were dosed daily with interleukin-1 receptor
antagonist protein (IRAP), by intraperitoneal injection, beginning on
day 14 after immunization, and continuing throughout the study.
The onset of arthritis was evaluated daily. Numbers in parentheses
are the number of arthritic mice at the end of the study. NT = not
tested.
t P < 0.01 versus saline controls.
.t P < 0.001 versus saline controls; P < 0.03 versus human serum
albumin (HSA) controls.
Table 3. Severity of collagen-induced arthritis in IRAP-treated
mice*
Treatment
group
Control
Saline vehicle
HSA (300 pglday)
IRAP
3 &day
30 PddaY
300 &day
Prednisolone (75 pglday)
Arthritis severity score,
mean t SD
Expt. 1
Expt. 2
4.4 2 1.7 (8)
2.7 ? 1.3t (9)
4.5
4.6
2.3 k 1.2f (7)
2.2 2 1.Of (8)
3.0 i 0 (1)
4.0
1.0
No arthritis
f 0.7
(6)
* 1.1 (5)
f
2.3 (3)
O§ (I)
No arthritis
NT
f
* Arthritis severity scores for each mouse were determined as
described in Materials and Methods. Numbers in parentheses are
the number of arthritic mice at the end of the study. See Table 1 for
definitions.
t P < 0.05 versus saline controls.
.t P < 0.01 versus saline controls.
I P < 0.05 versus both saline and HSA controls.
in the 2 control groups, and on days 32 and 36 in the 3
pg and 30 pg IRAP groups, respectively.
In the second study, the emergence of disease
was slower, with a mean onset at 55.5 days in the
saline controls, compared with 32.4 days seen previously. While the mean day of onset was delayed in
both the 3 pg and 30 pg daily IRAP groups, paralleling
the results from the first study, these differences did
not achieve statistical significance. The days on which
arthritis was first detected were, respectively, days 39
and 41 in the saline and HSA controls, and days 51 and
65 in the 3 pg and 30 pg IRAP groups. Since I M P
completely prevented arthritis at the 300 pglday dosage, no day of onset could be recorded.
Effect of IRAP on the severity of CIA. The effect
of IRAP treatment on the severity of CIA in affected
limbs is shown in Table 3. In the first experiment,
treatment with HSA reduced the severity index compared with saline vehicle treatment. However, this
decrease was not seen in the repeat study. Significant
reductions in disease severity were seen in the 3 pg
and 30 pg IRAP treatment groups compared with
saline vehicle, but not compared with HSA. In the
second study, IRAP at 30 pg significantly reduced the
severity score in the 1 arthritic animal compared with
either the saline or HSA control groups, and there was
no arthritis to assess in the 300 pg/day IRAP treatment
group.
No significant differences in mean peak paw
swelling were observed in the arthritic animals in any
of the IRAP treatment groups and the control animals.
IL-1 ANTAGONIST PROTEIN IN MURINE ARTHRITIS
Table 4. Serum levels of anti-type I1 collagen antibodies in IRAPtreated mice*
Optical density,
mean f SD
Treatment
group
Control
Saline vehicle
HSA (300 pglday)
IRAP
3 PgldaY
30 PgldaY
300 &day
Prednisolone (75 pglday)
Expt. 1
Expt. 2
2.98 f 0.38
2.79 t 0.59
1.00 f 0.12
0.94 2 0.19
2.74 t 0.49
2.49 2 0.537
2.10 f 0.51$
1.42 t 0.671
1.00 f 0.14
0.86 f 0.14t
0.71 2 0.081
NT
* Anti-type I1 collagen antibody levels were determined by enzymelinked immunosorbent assay of serum from each mouse, as described in Materials and Methods. Sera were obtained on day 48
(Expt. 1) and 42 (Expt. 2), and the optical densities in diluted sera
(1:200) from 10 animals per group were averaged. Values for day 42
sera (Expt. 2) were representative of day 56, day 70, and day 90
sera. See Table 1 for definitions.
t P < 0.05 versus saline controls.
$ P < 0.001 versus saline controls; P < 0.01 versus HSA controls.
J P < 0.001 versus saline controls and versus HSA controls.
No significant weight loss was seen in any of the
experimental groups, and IMP-treated animals remained in good physical condition throughout the
study (data not shown).
Effect of IRAP on the antibody response to type
I1 collagen. Levels of antibody to type I1 collagen were
measured in serum samples collected on day 48 and
day 42. Analysis of sera from individual mice, within
treatment groups, showed that antibody levels in nonarthritic mice were not significantly different from
those in arthritic mice. Serum values were therefore
averaged for all mice within a group (Table 4). No
1309
change in antibody level was seen in either of the 3 pg
IRAP groups (experiments 1 and 2). At 30 pglday,
IRAP caused a slight suppression of anticollagen antibody levels (P < 0.05) compared with saline but not
HSA. Treatment with 300 pg IRAP, however, significantly suppressed the anticollagen response in both
experiments, compared with the saline (P< 0.001 and
P < 0.001) and HSA (P < 0.01 and P < 0.001)
treatment groups. Serum anticollagen antibody levels
were more suppressed in the prednisolone-treated
mice than in the IRAP-treated mice.
Effect of IRAP treatment on AIA. The ability of
IRAP to suppress AIA in mice immunized with mBSA
was investigated using 3 separate treatment protocols.
In the first study, daily doses of 3, 30, or 300 pg of
IRAP were given, starting the day of initial immunization (day -21) until the day of intraarticular mBSA
challenge (day 0). This regimen failed to alter either the
pattern or the severity of the histopathology. The global
arthritis scores, based on inflammation, pannus growth,
cartilage damage, and bone resorption, were not significantly dif€erent between the IRAP treatment groups and
the saline or HSA treatment groups (Table 5).
Two subsequent studies were performed to
evaluate the effect of IRAP treatment during the
development of arthritis. Mice were dosed from the
day of secondary immunization (day -7), prior to knee
challenge, and subsequently for 14 days after the
induction of arthritis. Alternatively, dosing was
started on the day of intraarticular challenge (day 0)
and was continued until day 14. Neither regimen
resulted in suppression of the articular pathology
(Table 5).
Table 5. Effects of IRAP on mBSA-induced arthritis*
Treatment
group
Control
Saline vehicle
HSA (300 pglday)
IRAP
3 /&day
30 I d d a y
300 pglday
Histologic arthritis score,
mean t SD
Days -21 to 0
Days -7 to 14
Days 0 to 14
17.3 f 3.8
17.7 2 3.0 (+2)
15.5 t 2.5
NT
15.9 f 5.8
NT
17.2 t 2.8 (-1)
18.3 f 2.3 (+5)
17.8 2 3.5 (+3)
17.3 t 3.1 (+12)
16.3 2 3.4 (+5)
16.1 2 4.1 (+4)
16.8 ? 4.6 (+6)
17.8 f 4.9 (+5)
16.1 ? 4.1 ( + I )
* Groups of 10 C57BV6 mice were treated with interleukin-1 receptor antagonist protein (IRAP) for the
periods shown, relative to the induction of arthritis by intraarticular antigen challenge (methylated
bovine serum albumin [mBSA]) on day 0. On day 14, the injected knee was removed and examined
histologically. The severity of inflammation, pannus development, cartilage erosion, and bone erosion
was assessed as described in Materials and Methods, and a combined average score was calculated for
the group (maximum 20). Numbers in parentheses show the percentage of change in arthritis seventy
relative to the saline vehicle-treated group. HSA = human serum albumin; NT = not tested.
WOOLEY ET AL
1310
Effect of IRAP on spleen cell responses to mBSA
and serum anti-mBSA antibody levels. Spleen cells,
prepared from IRAP-treated and vehicle-treated arthritic mice, were stimulated in vitro with mBSA to
determine whether IRAP had an effect on antigenspecific cell proliferation. No response to mBSA was
seen using spleen cells obtained from normal, nonimmunized mice (Figure 1). Cells from arthritic mice
treated with vehicle, however, proliferated strongly in
a concentration-dependent manner. These responses
were significantly higher in mice given 3 or 30 pg of
IRAP (day -7 to day 14), while the response in mice
b
L
25000
A
06 2
Vehicle
lRAP3pg/day
lRAP3Opg/day
5
3
Serum Dilution (log 10)
20000
Figure 2. Effect of interleukin-1 receptor antagonist protein (IRAP)
on serum anti-methylated bovine serum albumin antibody (antimBSA Ab) 1gGl levels in arthritic mice. Levels of anti-mBSA IgGl
were measured by enzyme-linked immunosorbent assay of sera
from mice treated daily with IRAP beginning 7 days before and
continuing to 14 days after arthritis induction. Sera from all mice in
each group were pooled. Values are the mean absorbance obtained
from triplicate wells.
-;
g
15000
._
I
-z
c
0,
a
-
3
10000
5000
0
10”
-’, --.
”
I
.. . ,
-
loo
lo1
1o2
mBSA (pg/ml)
Figure 1. Spleen cell proliferation to methylated bovine serum
albumin (mBSA) from arthritic mice treated with interleukin- 1
receptor antagonist protein (IRAP). Antigen-induced arthritis was
provoked with mBSA in groups of 10 C57B1/6 mice, and in vitro
spleen cell proliferation to mBSA was assessed on day 14. Daily
treatment with saline vehicle or IRAP was begun 7 days before and
was continued to 14 days after arthritis induction. Spleen cells from
normal, nonimmunized mice were used as a negative control. Spleen
cells (5 x lO’/well) were incubated for 4 days and pulsed with
tritiated thymidine for the last 18 hours of culture. Values are the
mean 2 SD cpm from triplicate cultures. Mean & SD proliferation of
unstimulated cells was 83 2 18 for normal cells, 138 2 16 for
vehicle-treated cells, 232 2 34 for 3 pg IRAP-treated cells, 321 2 75
for 30 fig IRAP-treated cells, and 227 ? 70 for 300 pg IRAP-treated
cells.
given 300 pg was almost superimposable on that in
vehicle-treated animals.
Serum levels of IgG1, IgG2, IgG3, and IgM
anti-mBSA antibodies were quantitated by ELISA.
No consistent effect of IRAP on the level of any
anti-mBSA antibody isotype in any experiment was
seen. Serum IgGl anti-mBSA antibody levels from
mice dosed with IRAP from day -7 to 14 are shown in
Figure 2.
Anti-IRAP antibody response in CIA and AIA.
Individual sera from IRAP-treated mice with CIA
were collected on day 48, pooled, and analyzed for the
presence of anti-IRAP IgG. No significant levels of
antibody were detected in either the saline or HSA
control groups or in mice treated with prednisolone.
Significant levels, however, were present in all of the
IRAP treatment groups (Figure 3A). The strongest
antibody response was seen in the sera of CIA mice
given 3 pg of IRAP daily, which produced a halfmaximal absorbance reading at a dilution of 1:5,000.
Both the 30 and 300 pg IRAP group sera produced
half-maximal absorbance values at 1 :1,000 serum
dilutions and produced ELISA profiles similar to that
-
-
1311
IL-1 ANTAGONIST PROTEIN IN MURINE ARTHRITIS
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than those seen in collagen arthritic mice (Figure 3B).
Serum from normal C57BY6 mice or mBSA arthritic
mice treated with saline vehicle had no detectable
levels of anti-IRAP antibody. Antibody in the 3 pg
IRAP group was barely detectable at a 1:lO serum
dilution. Anti-IRAP antibody was detected in the 30
and 300 pg IRAP groups at 1:lO and 1:lOO serum
dilutions but not at 1: 1,000.
0.4
0.0
0
1
2
3
4
5
6
Serum Dilution (log 101
Figure 3. Levels of anti-interleukin-1 receptor antagonist protein
antibody (anti-IMP Ab) in mice with A, type I1 collagen-induced or
B, methylated bovine serum albumin-induced (mBSA) arthritis.
Sera from collagen arthritic DBA/1 LacJ mice treated with IRAP on
day 48 (experiment 1) and from mBSA arthritic C57BV6 mice treated
with IRAP beginning 7 days before and continuing to 14 days after
arthritis induction were pooled into groups, and an enzyme-linked
immunosorbent assay was used to determine circulating levels of
anti-IRAP antibody. Sera from mice deliberately immunized with
IRAP was used as a positive control. Values are the mean absorbance obtained from triplicate wells.
seen with a positive control serum from (BALBIcJ X
A/J)F1 mice immunized with IRAP.
Analyses of individual mouse sera from the 300
pg IRAP group revealed that 4 mice which had no
detectable CIA had more serum anti-IRAP antibody
than did the 1 mouse which developed arthritis. Thus,
the failure of IRAP to prevent arthritis in this animal
was not associated with a high titer of neutralizing
antibody. Serum anti-IRAP antibody levels in mBSA
arthritic mice dosed from day -7 to day 14 were lower
In the present studies, we examined the effects
of a human IL-1 receptor antagonist protein on both
type I1 collagen-induced and mBSA-induced arthritis
in mice. This recombinant 17-kd U937 cell protein is
identical to that originally cloned by Eisenberg et a1
(IL-lra) from human peripheral blood monocytes (18).
Four similar proteins (22-26 kd), which appear to be
N-linked glycosylation isoforms, have also been purified from the supernatant of phorbol myristate
acetate-differentiated THP-1 cells (26). All of these
proteins compete with IL-lpfor binding to IL-1 receptors on T cells, with Ki values of 0.1-0.6 nM, but do
not possess IL-1 agonist activity in functional bioassays (19,20). Importantly, IRAP/IL-Ira has functional
IL-1 antagonist activity in vivo. IRAP inhibits IL-1induced elevation of serum corticosterone in mice with
an ID,, (50% inhibitory dose) of 0.3-3 pg/mouse (19),
and IL- Ira will inhibit IL-1-induced endotoxin shock
in mice (27) and IL-1-induced neutrophilia in mice
following administration of IL- 1, lipopolysaccharide,
or proteose-peptone (28).
In our studies, 300 pg of IRAP/day, given
intraperitoneally , profoundly suppressed the incidence
of CIA by 88% and loo%, respectively. In one study,
in which the arthritis had a slower onset and affected a
smaller number of mice, 30 pg/day also produced a
significant reduction in incidence, suggesting that
milder CIA may be more amenable to suppression
with lower doses of IRAP. Although IRAP treatment
delayed the onset of arthritis at 3 pg/day and 30
pg/day, these differences were not large (-5-10 days),
and the 1 mouse with arthritis in the 300 pg group
developed disease at the same time as the control
mice. Similarly, disease severity in mice which developed CIA while receiving IRAP was suppressed in
some cases, but was complicated by an unexpected
suppressive effect of HSA in 1 experiment. Thus,
while IRAP clearly decreased the incidence of arthritis, the onset and seventy of CIA in mice “resistant”
to IRAP was suppressed in some but not all mice.
1312
Precisely how antagonism of the biological effect of IL-1 leads to suppression of CIA is unclear.
One possibility is that IL-1 is critical for the development of immunity to type I1 collagen. Although IRAP
treatment reduced serum levels of antibody to type 11
collagen, it seems unlikely that these relatively small
reductions, by themselves, would be sufficient to
suppress the arthritis. Since administration of IL- 1 to
mice with CIA has been shown to both accelerate and
exacerbate the arthritis (11,12), an additional possibility is that IL-1 plays a direct role in joint inflammation
and destruction, which can be attenuated by IRAP.
In contrast to the suppressive effects of IRAP
on CIA, we were unable to inhibit mBSA-induced
arthritis. Treatment during the immunization phase
had no effect on AIA, suggesting that IRAP did not
suppress the development of immunity to mBSA.
IRAP treatment from the day of antigen challenge or 1
week prior to arthritis induction also had no effect on
the resulting histopathology . These observations correlated with a lack of inhibition of both in vitro
antigen-specific spleen cell proliferation and serum
levels of anti-mBSA antibodies. However, the mBSA
arthritis model can be suppressed by steroids, azathioprine, and methotrexate as well as the immunosuppressive agents cyclosporin A and FK-506 (29,30),
suggesting that it is a useful experimental model of
human RA.
IL-lra at 2-3 mg/kg has been shown to inhibit
joint swelling in rat streptococcal cell wall-induced
arthritis reactivated by challenge with peptidoglycanpolysaccharide polymers (15). This demonstrates that
endogenous IL-1 plays a role in the early inflammatory
response in this model. Anti-IL-1 antiserum prevented
the suppression of cartilage proteoglycan synthesis
associated with the early phase of AIA in mice, but
had only a modest effect on joint inflammation (16).
These observations suggest that IL-1 plays an important role in the early inflammatory events in arthritis.
Since an important pathogenic mechanism in CIA is
the ingress of type I1 collagen-reactive T cells into
joints, inhibition of early inflammation mediated by
IL-1 may help protect against CIA. This is supported
by the observation that nonsteroidal antiinflammatory
drugs inhibit the exacerbation of CIA by exogenous
IL-1 (31). Since mBSA is injected directly into sensitized joints in AIA and is accompanied by an early
aggressive pathology, this model may be less sensitive
to IL-1 antagonism. Alternatively, it may be that
chronic AIA is not dependent on IL-1, and other
cytokines are responsible for the immunopathogenesis
WOOLEY ET AL
of the arthritis. In rats, intraarticular injection of IL-1
resulted in inhibition of mBSA-induced arthritis (32),
suggesting that under certain conditions, IL-1 may
have an antiinflammatory effect, possibly via the induction of endogenous corticosterone (33).
The difference in sensitivity between the effects
of IRAP in the CIA and AIA models may, however, be
due to other possibilities. If AIA generates more
endogenous IL-1 than CIA, then concentrations of
IRAP higher than those we tested might be required to
inhibit AIA. It seems unlikely that the pharmacokinetics of the protein would be different in DBN l and
C57BV6 mice, and IL-lra has been shown to be an
effective inhibitor of endotoxin shock in C57B1/6 mice
(27). The failure of IRAP to inhibit mBSA-induced
arthritis could be due to the development of neutralizing antibodies to IRAP. However, this seems unlikely. While we did not directly measure neutralizing
antibodies, anti-IRAP IgG was present in higher titers
in mice with CIA than in mice with AIA. Furthermore,
300 pg/day of IRAP suppressed CIA despite anti-IRAP
antibody titers comparable to levels of antibody produced by mice deliberately immunized with IRAP.
Last, the level of anti-IRAP antibody in individual
mice did not correlate with the presence or absence of
collagen-induced arthritis.
Recently, IRAP has been immunolocalized in
perivascular areas of the sublining layer of rheumatoid
synovial tissue, and IRAP gene expression was identified in some, but not all, synovial tissue samples (34).
Neutrophils, as well as monocytes, produce IL-lrd
IRAP, and substantial levels have been measured in
RA synovial fluids (35). Several reports have described the presence of IL-1 inhibitors in RA serum,
synovial fluids, and cell culture supernatants which
interfere with the measurement of IL-1 in bioassays
(3,4,36). Whether these inhibitors are the same as, or
are closely related to, IL-1 receptor antagonist proteins, remains to be determined. A binding protein
specific for IL-1p has been found in RA serum and
synovial fluid (37); this may be a soluble form of the
type I1 IL-1 receptor identified in Raji B cell supernatants (38). Two other IL-1 inhibitors have been described: endogenously produced anti-IL- 1a antibody
(39) and an IL-la-specific binding protein purified
from RA synovial fluid (40) which may also be an
antibody or antibody fragment. Measurement of these
IL-1 antagonists and binding proteins will help determine whether imbalances between IL-1 and its endogenous inhibitors contribute to the pathology of inflammatory diseases such as rheumatoid arthritis.
IL-1 ANTAGONIST PROTEIN IN MURINE ARTHRITIS
ACKNOWLEDGMENTS
We thank K. Franz for preparing the histologic
specimens and Drs. D. Tracey, D. Carter, and M. Bienkowski for the IRAP.
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