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Effects of antiinflammatory agents on the acute response of immune synovitis in rabbits.

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937
EFFECTS OF ANTIINFLAMMATORY
AGENTS ON THE ACUTE RESPONSEOF
IMMUNE SYNOVITIS IN RABBITS
MARVIN B. GOLDLUST. LOIS C. RICH. and THOMAS W. HARRITY
Immune synovitis in rabbits was investigated as a
potential in vivo model for evaluating new antiinflammatory agents. Antigen-induced increases in knee width
as well as P-glucuronidase and acid phosphatase activities in exudates were observed. Histologically, polymorphonuclear leukocytes appeared within hours in synovial tissues and reached maximum infiltration at about 24
hours. Subsequently, mononuclear cells, including plasma
cells, appeared. The 6-hour Arthus-like phase of synovitis
can be depressed by some antiinflammatory agents, colchicine and steroids being particularly effective. It is suggested that this model can be utilized to define potentially
more effective antiinflammatory drugs.
Immunologically-induced synovitis in rabbits ( 1 )
has been investigated in an attempt to develop new
models in vivo which can define more effective antirheumatic agents. I n the present studies bovine serum albumin (BSA) has been utilized as the antigen. This synovitis model demonstrates many of the synovial fluid and
tissue characteristics observed in rheumatoid synovitis.
Parallels between rheumatoid arthritis (RA) and this
experimental model include deposition of immune comFrom the Immunology Section, Department of Pharmacology. Squibb Institute for Medical Research, Princeton, New Jersey.
Presented in part at the VI Pan-American Congress on Rheumatic Diseases. Toronto, Canada. June 16-21, 1974.
Marvin B. Goldlust. Ph.D.: Senior Research Investigator:
Lois C. Rich. M.S.: Research Associate: Thomas W. Harrity, M.S.:
Assistant Research Investigator.
Address reprint requests t o Marvin B. Goldlust, Ph.D., P. 0.
Box 4000. Princeton, New Jersey 08540.
Submitted for publication June 9, 1976: accepted December
13. 1976.
Arthritis and Rheumatism, Vol. 20, No. 4 (May 1977)
plexes and complement in avascular tissues such as cartilage (2.3), infiltration of synovial tissue with macrophages, lymphocytes, and plasma cells (4-7), and the
demonstration of immunoglobulin synthesis by synovial
tissue (7,8). I n addition, the inflammatory exudates in
the joints of rheumatoid patients and of animals with
immune synovitis contain predominantly polymorphonuclear leukocytes (PMNs) (1,5,7).
Other workers have used synovitis models to examine the effects of antiinflammatory agents. In these
studies synovitis was induced by intraarticular injections
of a variety of agents, including urate crystals (9,10),
antigen ( 1 1-13), and antiserum (14-16). In the present
studies we investigated the effects of antiinflammatory
agents on antigen-induced synovitis by using a biochemical procedure @-glucuronidase) to measure exudate
PMN accumulation. Cellular infiltration into the synovial tissue was also measured histologically. Because of
the similarities between RA and immune synovitis, and
because of the potential for experimentally investigating
the transition from acute to chronic inflammation, we
began our studies on the acute phase of this model.
MATERIALS AND METHODS
Sensitization of Rabbits and Induction of Synovitis.
Male New Zealand white rabbits (1.5-2 kg) were acquired
from Davidson Mill Farms, North Brunswick, NJ. They were
housed singly in standard wire-bottom cages and allowed water and feed (Ralston Purina Rabbit WO or Rockland Teklad
Rabbit Ration) ad libitum. Rabbits were sensitized to BSA as
follows. An emulsion of equal volumes of BSA (10 mg/ml in
GOLDLUST ET AL
938
saline) and Freund's complete adjuvant was made, and 2 mg
of BSA in adjuvant were injected into the pads of each hind
foot. Two or 3 weeks later, animals were injected intradermally on the back with 250pg of BSA. The skin responses were usually assessed 24 hours later, just before implementing an experiment. Animals not responding were discarded,
but discarding was only rarely necessary. Sensitized rabbits
were challenged intraarticularly in the shaved right knees,
usually with either 0.5 or 1 mg of BSA in 0.1 ml pyrogen-free
saline (PFS). The left knees received PFS alone. To ensure
sterility after intraarticular injection, all materials were filtered
through Swinnex,"' (Millipore) 0.2 p filters before injection.
Standard microbiologic tests were performed on representative samples of tissues and exudates and they indicated a sterile
inflammation.
Measurement of Synovitis. At varying times after intraarticular challenge with BSA, animals were sacrificed by
intracardial injections of Lethol" (sodium butabarbital and sodium pentabarbital, Elkins-Sinn, Cherry Hill, NJ), and the joint
capsules were excised. These tissues were fixed in 10%formalin
and slides for histologic evaluation were prepared (Histological Enterprises, Fort Washington, PA). Five to 6 pm sections
through the synovial membrane and underlying tissue were
stained with hematoxylin and eosin. At least two tissue sections per joint were evaluated. These sections were examined
for infiltrating cells in the blind by a single investigator using a
0 to 4 subjective scoring system, in increments of 0.5, as
described below. With a stage micrometer to standardize the
eyepiece grid, actual PMN counts of four fields (0.065 mm')
per section under 400X magnification were made on more
than 50 tissue samples. Accurate overall counts for each section were difficult because areas ranging from minor to extensive PMN infiltration occurred. Thus the data only roughly
suggested the range of PMN densities presented in Table 1.
The histology scores indicated in the present studies
were derived from an overall assessment of the cell types after
examination of the tissue sections under IOOX and 400X total
magnification, and then scoring. Although the two scoring
methods were compared. the overall assessment was felt to be
more indicative of the general state of the tissue and was
considerably less time consuming than was cell counting. Saline-injected knees virtually always gave scores of 0. The average PMN scores for vehicle-treated groups were between 2 and
3.5.
Fluid in the joint was removed with a disposable glass
pipette and placed at 0°C. The joint space was then flushed
with 0.5 ml of cold PFS dropwise with continuous aspirating
of the fluid with a disposable pipette. The wash was added to
the original exudate. Samples were then stored at -75°C for
less than 1 week before evaluation. T o disrupt cells in preparation for enzyme assay, Triton X-I00 was added in a small
Table 1. Densiry o] P M N Injiltration in a
0-4 Subjective Scoring System
Score
PM Ns/m mz
<50
150-400
350- 1200
I OOO- 1 700
1600-4300
volume ( I : 10 v/v) to the thawed samples of exudate flushes to
give a final concentration of 0.2%. Preliminary experiments
had indicated that this was about the maximum concentration
that could be used without inhibiting 0-glucuronidase. The
samples with Triton were then incubated for at least 15 minutes at O C and centrifuged a t I 1 0 0 X g for 10 minutes before
the supernatants were assayed. When selected exudate samples were examined microscopically, both debris and apparently intact cells were observed. All cells took up Trypan
Blue only after Triton treatment and released >85% of the
total enzyme.
Enzyme values (optical densities [OD] at 550 nm) for
exudate flushes from PFS-injected knees were usually considerably less than 5% of the values for the BSA-injected knees.
Saline values were subtracted from the BSA values before
analysis of the data. The extent of inhibition of exudate formation by compounds was calculated by comparing the average
O D of joint flushes of compound-treated animals to those of
control animals.
A modification of the above method of evaluation was
also used for some experiments. In this method the O D values
were corrected for the volumes of exudate Rushes recovered.
These values were then converted into total numbers of PMN
equivalents per joint by using the graph in Figure 2, which
gives the number of PMNs per OD. In equation form,
ml exudate X OD X PMNs/OD
Total PMNs/joint
=
ml exudate assayed
I n the experiments to be discussed, the range of concentrations of PMN equivalents observed in control Rushes
was 8.000 to 50.000 per mms. or 4 X 10' to 5 X IO'/joint.
Knee widths were measured with a caliper. Measurements were made just before and at various time points after
intraarticular challenge with antigen or PFS. The data presented are corrected for zero time differences in width between
right and left knees.
Leukocyte counts in exudate flushes (Figure 1 ) were
made as follows. Samples were centrifuged for 10 minutes at
260 X g and resuspended to the original volume in PFS. A
dilution was then made in 1% normal rabbit serum containing
Trypan Blue so that 100-200 cells per sample were counted on
a Bright Line hemacytometer (American Optical, Buffalo,
N Y ) . Differential leukocyte counts were made on air-dried
smears of exudates that were treated with Hemal Blood Film
Stain (Hemal Stain Co, Danbury, CT). One hundred cells per
smear were counted.
Statistical Evaluation of Data. Significance was calculated by the Wilcoxon rank sum test utilizing the table indicated in Dixon and Massey (17). An experimental result was
considered significant if P was less than or equal to 0.05.
Enzyme Assays. Samples of 0. I or 0.2 ml were analyzed
for 0-glucuronidase activity by the procedure of Talalay et a/
(18). The reaction mixtures consisted of 0.1 ml (0.53 mg)
phenolphthalein glucuronic acid, sodium salt (Sigma Chemical
Co. St. Louis. MO). enzyme sample, and 0.4 M sodium acetate
buffer at pH 4.6, to a final volume of 1 .O ml. After 18 hours of
incubation at 37°C with shaking, 3.0 ml of 0.4 M glycine
buffer at pH 10.4 were added, and the reaction tubes were
centrifuged at 1 100 X g for 10 minutes. The optical densities of
the supernatants at 550 nm were recorded. Sixteen micrograms
of P-glucuronidase standard (7.5 units/g) (Worthington Bio-
ANTIINFLAMMATORY AGENTS AND I M M U N E SYNOVITIS
L
a
[
I
I
I
I
-E,
0
8
-8#
a
2
*
I
* I
I
8
a
( p = <.001)
0.8 -
a
0.6 -
3
3
0
-
1
1
a
a
r = 0.763
1.0
1
1
1.2 -
939
*
a
a
a
a
a
0.4
-
a
a’
0.2 -
(3
ch
3 1
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I
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PMN
X I O ~
,1713
Fig I . C‘orrelarion between P M N s and @-glucuronidase in Pushes OJ joinrs obrained 6 hours aJier inrraarricular challenge wirh 0.5 tng of BSA inro each o f 2 3 rabbit knees. A correlation coeficienr ( r ) of 0.763
P < 0.001) nos/ound.
chemical Corp, Freehold, N J ) per assay tube produced an
optical density in the range of 0.8 to 1.2.
Acid phosphatase was assayed by the method of Andersch and Szczypinski (19). Samples of 0.1 ml were added to
tubes containing 0.8 ml of 0.09 M sodium citrate buffer at pH
4.8 and 0.1 ml (5.6 mg) ofp-nitrophenylphosphate.disodium.
5 I / 2 H,O (Worthington). Tubes were incubated with shaking
for I hour at 37°C. Three milliliters of 0.1 M NaOH were then
added. followed by centrifugation for 10 minutes at 1100 X g.
The optical densities of the supernatants were recorded at 410
nm. Wheatgerm acid phosphatase (Worthington, 0. I6
units/mg) served as a reference.
Preparation of Peritoneal Exudate. A rabbit was infused intraperitoneally with 500 ml of I % shellfish .glycogen
(Mann Research, New York, N Y ) in PFS. Eighteen hours
later the peritoneal exudate (>95% PMNs) was collected into
Tyrode’s solution (Grand Island Biological Co, Grand Island,
NY). Cells were then washed and suspended to a known
concentration in PFS. Unlike synovial exudate PMNs, these
peritoneal cells exhibited little clumping.
Additional Materials. Isotonic pyrogen-free saline
(PFS) was purchased from Cutter Laboratories, Berkeley, CA,
or Abbott Laboratories, North Chicago, IL. Suspending vehicle (SV) was prepared as a sterile solution of 5 mg sodium
carboxymethylcellulose. 4 mg Tween 80. 7 mg NaCI, and 9 mg
benzyl alcohol per ml of distilled water.
Difco Freund’s complete adjuvant with M bufyricum
was used. Bovine albumin, crystalline (BSA), was acquired
from Nutritional Biochemicals Co. St. Louis, MO. Triton X100 was purchased from Schwartz/Mann, Orangeburg, NY.
The following agents (with suppliers in parentheses)
were also tested: phenylbutazone (Geigy): acetylsalicylic acid
(Monsanto): indomethacin (Merck): niflumic acid, triamcinolone acetonide. and its 2 I-dipotassium phosphate ester derivative (Squibb): prednisolone and methylprednisolone (Upjohn).
These agents were prepared for administration to rabbits by first suspending and homogenizing compounds i n SV.
Nine volumes of PFS were then added with additional dispersion by means of glass-glass homogenizers or, in a few
studies, by a Polytron” homogenizer (Brinkmann Instruments
Inc. Westbury. NY). The compounds were then administered
by the intraperitoneal or oral route 0.5 or 1 hour. respectively,
before intraarticular challenge with BSA. All compounds were
administered in 1 ml/kg, except phenylbutazone and acetylsalicylic acid, which were administered in 2 ml/kg.
R ESU LTS
8-Glucuronidase Correlation with PMNs in Joint
Effusions. P M N s in j o i n t effusions in t h e immune synovitis model aggregate so t h a t a c c u r a t e cell c o u n t s a r e
difficult. To analyze t h e a c u t e exudative response, t h e
activity of lysosomal 8-glucuronidase w a s measured.
PMN c o u n t s a n d 8-glucuronidase activity were determined in t h e same j o i n t effusions t o find whether a
correlation existed between these t w o parameters. A
positive correlation in 6-hour exudates was observed
(Figure I ) . Based on a total of 23 knees, each injected
with 0.5 mg o f BSA, t h e correlation coefficient w a s
found t o be 0.763 (P < 0.001). Thus t h e use o f 8glucuronidase activity t o indicate t h e presence of exu d a t e PMNs provided a simpler, more practical, a n d
probably more a c c u r a t e p r o c e d u r e t h a n c o u n t i n g
PMNs.
A reference ‘curve w a s generated (Figure 2) to
relate t h e enzyme levels in j o i n t exudates t o PMN concentrations in these exudates without having t o count
940
GOLDLUST ET A L
I
4.0
I
1
I
I
1
1
1
4
5:
10
5
w
lx
P
3.0
v,
3
*
0
V
z
LYMPHOCYTES
ul
>
w
n
(3
5
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Q
0
I-
2
E
2.0
I-
8I
2
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I
w
v,
a
ez
0
a
0.5
I .o
2
40
0
3
0.5
(3
80
120
HOURS
160
2
4
6
8
WEEKS
10
TIME AFTER INTRA-ARTICULAR BSA CHALLENGE
I
P
1
2
3
4
5
6
7
8
9
NO. PERITONEAL EXUDATE PMNs /ASSAY TUBE
x 106
Fig 2. B-glucuronidase actiuity as a funcrion of the number of rabbit
perironeal exudate P M N s in the assay.
PMNs for each experiment. The source of the PMNs
used in this experiment was an 18-hour rabbit peritoneal
exudate, and it was assumed that the enzyme content in
these cells was similar to that in joint exudate cells.
Enzyme assays were performed on Triton X-100-disrupted cells. As Figure 2 shows, a linear relationship was
found up to about 3 X lo6 PMNs/tube. Utilizing these
data and the results found with a commercial preparation of /3-glucuronidase, we calculated that there is
about 1.6 X
pg of enzyme per PMN.
Table 2. Acute Response in Rabbit Synooitis at 6 Hours: Effect of
Varying Intraarticular Doses of BSA
BSA*
(pg)
1000$
500
100
10
I
0.1
Average PMN Equivalents per mms of
Exudate Flush f SD
1.5 f
1.5 f
1.6f
4.0 f
1.8f
12.5 f
0.5 X 10'
0.7 X 10'
0.7Xl0'
0.2 X IOs6
0.2 X loSg
0.04 X 1025
Average PMN
Histology Score
of Synoviurn (range)t
2.8 (2-4)
3.5 (3-4)
2.8 (2.5-3)
2.0(1-3)
I . 3 (1-2)
0.5 (0-1)
* Four rabbits per group: right knees received 0.1 rnl of BSA. left
knees received 0.1 rnl saline.
t PMNs scored 0 to 4. Values are for BSA-injected knees: those for
saline-injected (left) knees were 0.
$ Separate experiment; 5 rabbits per group.
pip < 0.05 vs group receiving the next highest BSA dose.
Fig 3. The appearance oj'injiltraring cells in synovial tissue from rabbits
challenged with I ing oJ' BSA inrraarricularly. Seven rabbits per rime
point were eualuated. Other mononuclear cells refers lo nionocyres and
rnucrophages, (*) indicares rhar P < 0.05 and (**) that P < 0.01 j o r a
group 1;s the group at the iinmediately preceding time point. P > 0.05
unless orherwise indicared.
Dose Response of Intraarticular BSA. Varying
quantities of BSA were injected intraarticularly to determine the sensitivity of the acute synovitis reaction to
antigen. The results in Table 2 demonstrate that the
exudative response, measured both biochemically and
histologically, plateaus at a dose of 100-1000 pg of BSA
per joint. This level may depend somewhat on the level
of sensitivity of a particular group of rabbits.
Evolution of Synovitis. Figure 3 indicates the time
of appearance of various cell types in the synovial tissue
following intraarticular challenge with 1 mg of BSA.
Groups of 7 rabbits were sacrificed at various times, and
the histology of the synovial tissues was evaluated. The
acute response was characterized by a peak of PMNs at
24 hours (range of scores, 3-4). Lymphocytes peaked at
72 hours (range of scores, 2-4). Monocytes and macrophages reached maximum infiltration by 1 week (range
of scores, 1-3 over the period of 1 week to 2 months).
Plasma cells increased over a longer period of time to a
maximum at 4-5 weeks (range of scores, 3-4 at 1
month). All cell types, except the PMNs, maintained
relatively constant or increasing levels in the synovial
tissue up to the last time point investigated, which was 2
months. Figure 3 does not indicate the other changes
observed in the synovium, such as increased vascularity,
lining layer cell hypertrophy, and hyperplasia, increased
fibrosis, and the occurrence of foci of lymphocytes and
ANTIINFLAMMATORY AGENTS A N D I M M U N E SYNOVITIS
94 1
Fig 4A. Appearance of'synouial tissue 6 hours after intraarricular challenge with 0.5 mg BSA. Note the numerous P M N s and the hypertrophy
oJ synouiocyres ( H di E stain, original magnification X 110).
Fig 48. Normal synovial tissue front the contralateral saline-injected
knee oJ'the rabbir in Figure 4A. ( H & E stain, original magnification X
plasma cells characteristic of this synovitis model (4-6).
Figure 4A depicts a histologic section of synovial
tissue from a knee 6 hours after challenge with 0.5 mg of
BSA. The major characteristic at this time is the influx
of PMNs. The single layer of synoviocytes has begun to
show hypertrophy, and edema can be observed. Figure
4 8 shows the 6-hour saline-injected, contralateral knee
that is normal in appearance. Figure 5 shows a histologic section of a chronically inflamed synovium removed from a knee 1 month after challenge with 1 mg of
BSA. Lymphocytes and plasma cells are readily seen.
The saline-injected contralateral knee, which is not
shown, was essentially normal. Erosions of cartilage
were not grossly observed in these studies.
For comparative purposes, /3-glucuronidase was
also assayed in joint flushes collected over the 2-month
period of the experiment indicated in Figure 3. Activity
apparently peaked at 24 hours (Figure 6), although no
statistically significant differences were found between
groups over the I-day to 1-week period. By 2 months
little enzyme was present. Figure 6 also records increases
in knee width that coincided with the appearance of
exudate /3-glucuronidase. The average maximum increase in width due to BSA was about 2 mm.
In a separate experiment, acid phsophatase as
well as /3-glucuronidase were evaluated in the same exudate samples before freezing. Figure 7 shows that the
appearance of these enzymes coincided over the 5-day
examination period.
Effects of Steroidal Antiinflammatory Agents on
Acute Synovitis. Table 3 lists the inhibitory effects of
four steroids. Significant decreases of exudate were dem-
1101.
942
GOLDLUST ET AL
P M N s . In 6 of the 20 assays, exudates were evaluated by
utilizing enzyme O D values directly and by calculating
total P M N s per joint as indicated in MATERIALS
AND
METHODS.These two methods showed excellent agreement.
Four other nonsteroidal agents have been evaluated after intraperitoneal administration in a number
of assays. Table 5 lists the data for indomethacin,
phenylbutazone, acetylsalicylic acid, and colchicine. The
variability of responses with the first three agents makes
it difficult to determine their potencies. However colchicine proved to be an extremely effective agent against
both exudate formation and P M N infiltration into synovial tissue.
Data on the steroids and nonsteroids indicate
that of 25 treatment groups analyzed for exudate by the
two methods described, 23 groups showed comparable
levels of inhibition and statistical significance by both
procedures. This result indicates that the observed variability in drug effects was most likely attributable to
factors other than the method of analysis, such as differences in the level of sensitization and inflammatory responses of individual animals.
DISCUSSION
Fig 5. Appearance oj’synouial tissue I month aJter intraarticular challenge with I nig BSA. Lymphocytes and plasnia cells can be seen ( H & E
stain. original tnagnijication X 1101.
onstrated by all agents when administered at low doses
by both the intraperitoneal and oral routes. The two
methods of exudate analysis were comparable. Variable
effects by these steroids were observed for P M N infiltration of synovial tissues.
Effects of Nonsteroidal Antiinflammatory Agents
on Acute Synovitis. Five nonsteroidal agents were evaluated in acute synovitis after intraperitoneal administration. Table 4 summarizes the results of 20 experiments
showing the effect of niflumic acid at 150 mg/kg. Fifteen
of 20 assays indicated significant depression of exudate
(P I0.05),the average being 5 I % for all assays. Fifteen
of 20 assays indicated decreased P M N infiltration into
synovial tissue with an average of 35% for all assays.
Two of those assays indicating nonsignificance of effects
on exudate were the same as those indicating nonsignificance of effects on membrane infiltration by
Our observations of the characteristics of immune synovitis generally agree with those of others
(1,4,5,11,20). There are, however, some differences.
Blackham et a1 ( I 1 ) found significant numbers of leukocytes in the synovial space 46 days after antigen challenge. Our observations (Figure 6) indicated that /3glucuronidase. representing PMNs, is not present in
significant amounts in the synovial space at 1 and 2
months. We also did not observe secondary increases in
knee widths at 1-2 months (Figure 6), but we examined
only two time points during this period. In addition, we
did not demonstrate significant numbers of cells other
than P M N s in exudates as others have reported
(5,11,20). These differences may be due to variations in
levels of sensitization of rabbits as well as to differences
in doses of intraarticular antigen.
I n the present studies, analyses of BSA antigeninduced synovial exudation (measured by the appearance of P-glucuronidase) and of P M N infiltration into
synovial tissue were utilized in an attempt to determine
effects of antiinflammatory agents. The steroidal agents,
triamcinolone acetonide and its soluble phosphate ester
derivative, prednisolone and methylprednisolone, all
significantly depressed exudate within 6 hours of intraarticular antigen challenge.
ANTIINFLAMMATORY AGENTS A N D IMMUNE SYNOVITIS
943
1000
g
-a
0
800
600
5
c
.0
7
\
Q
400
200
4c
Ea
-g
Q
(3
-c
I
3
5
7
9
II
DAYS
13
4
7
10
01
d
WEEKS
TIME AFTER INTRA-ARTICULAR BSA CHALLENGE
Fig 6. The appearance ofj'oint exudate (measured by 8-glucuronidase)(@), and the change in knee width ( 0 )
with iirtte aJrrr itiiraarticular BSA challenge. The exudates were from rabbits whose synovial tissues were
also rcaluared (See Figure 3). The enzyme results are average values of6-7 rabbits per time point and were
corrected for calues of 'saline-injectedjoints. The knee widths o j l 3 rabbits were sequentially evaluated up to I
uioiiih, and 7 )+'eri>
juriher evaluated at 2 months. The bars indicate the standard error OJ the mean; ( * * )
indit~uresihar P < 0.01 for a group us rhe group at the immediately preceding time point. P > 0.05 unless
orlier\r.ise itidicated.
The potencies of the nonsteroidal agents, with
the exceptions of niflumic acid and colchicine, were less
clear. We previously reported that indomethacin,
phenylbutazone, and acetylsalicylic acid possessed some
antiinflammatory activity in this synovitis model (21).
Additional replicate experiments presented here indicated a substantial variability in the observation of
I
700
v)
3
LL
+
z
4
500
J
W
300
?N
z
W
0,
a
100
24
48
5
HOURS
DAYS
TIME AFTER INTRA-ARTICULAR BSA CHALLENGE
Fig 7. C.ointidetiral appearance oj8-glucuronidase and acid phosphatase
uc~iiciiii~.c
in ,joint e.wdaies as a junction oJ time ajier inrraarticular
dialleiige with 0.5 iiig of B S A . Each value is theaverage oJ7animals and
nax correcied jor ualues oj saline-injected joints. The bars indicare the
siandurd error oJ'ihe niean; (*) indicates that P < 0.05 and ( * * ) that P <
0.01 ,/or a group L'S ihe group at the immediately preceding time point. P
> 0.05 U I I I ~ . F Soihrrwise iiidicaied.
6
statistically significant activity for these agents. One possible reason for this variability is that the intra-group
variations in inflammatory responses in untreated
groups is large and may partly reflect the fact that we
were using actively sensitized animals. I n fact, using a
reversed passive Arthus skin reaction (RPA) in rabbits
(22). we were able to demonstrate significant reductions
in edema after administration of the same doses of four
nonsteroidal agents used in the present studies. I t should
be remembered, however, that we are comparing skin
site edema with intraarticular PMN accumulation. Also,
the methodology involving measurement of each animal's pretreatment response in the RPA may allow for
more consistent results.
I t is also possible, as in the case with niflumic
acid (Table 4), that after performing enough assays, the
actual potencies of the other nonsteroidal agents could
be more clearly defined. The niflumic acid data d o indicate large variations in inhibition (range 15 to 81% for
exudate PMNs, Table 4). although standard errors are
small.
Observations of significant drug effects on the
extent of PMN infiltration into synovial tissues were
inconsistent. Except for niflumic acid, with which we
have performed many assays, the effects on PMN infiltration into tissue are unclear for the agents investigated. However the experiments with colchicine
and methylprednisolone indicated impressive decreases.
A possible reason for these inconsistencies may be the
relatively greater sensitivity and fewer technical prob-
GOLDLUST ET AL
Table3. Eflectsof Steroids on Ihe Acute Responseat 6 Hours in the Rabbit Synooitis Test
Group Size
Compound*
Triamcinolone
acetonide (TA)
Expt
Treated, Control
Route
I
5,9
479
6,6
6,6
799
6,6
739
66
799
6,6
.6,8
6,8
iP
iP
iP
iP
2
3
4
I
2
TA phosphate
Prednisolone
1
Methylprednisolone
2
I
2
Dose
(mg/kg)
2
0.2
0.2
1
1
1
PO
iP
PO
iP
PO
iP
PO
PO
PO
68
Percent Decreaset
of Exudate
I
I
I
1
1
0.1
0.01
OD
85
53
69
66
74
63
57
64
81
75
55
46
34
(<O.OOl)$
(0.001)
(0.008)
(0.001)
(<O.OOI)
(0.002)
Total PMNs
75
-
Percent Decrease
of PMN
Histology Score
24 (0.5)$
0
(0.001)
(0.006)
(0.002)
69 (0.002)
78 (0.001)
(<O.OOl)
(0.001)
(0.02)
(0.05)
(0.1)
82
62
53
41
-
(0.001)
(0.006)
(0.04)
(0.05)
26
37
46
53
23
0
23
67
41
38
24
(0.1)
(0.006)
(0.003)
(0.01)
(0.2)
(0.05)
(0.001)
(0.02)
(0.03)
(0.02)
* Compounds were administered
by the intraperitoneal (ip) or oral (PO) route 0.5 or 1 hour, respectively, before intraarticular challenge with 0.5
mg BSA.
t OD refers to calculations based on 8-glucuionidase absorbance (OD550) values. Total PMNs refers to calculations based on total enzyme (i.e..
PMN equivalents) recovered per joint.
$ p.
lems of the enzyme assay of exudate compared with
histologic scoring. Although the subjective nature of the
tissue evaluation may contribute to the inconsistency of
the data, a greater problem is suggested by the differences in magnitude of the inflammatory responses of
individual animals as manifested by PMN exudate formation.
Reports have appeared describing the effects of a
variety of therapeutic agents in synovitis models in rabbits and guinea pigs ( I 1-16). The parameters used to
assess synovitis in these studies included superficial
Table 4. Summary of Nij7umic Acid Eflects on Acute Response
at 6 Hours in the Rabbit Synovitis Test
~
~~
Parameter
Measured
~~
~
Exudate,
Tissue
histology
No. of
Experiments
~~
20
20
Average
Percent
Decrease
f SEM
Percent
Range
No. of
Tests
( P 20.05)
~~
51 f 4
35 f 3
15-8 I
11-60
15t
1st
* Exudate measurements were made by using the a-glucuronidase O D
values without correcting for exudate volumes recovered.
? O f the 5 tests that yielded nonsignificant data, t w o had P values
of > 0.05 for both parameters.
swelling, gross appearance of joints, exudate analyses,
and tissue histology. These studies suggest that the observed effectiveness of an agent may vary, depending
upon the dose and route of administration and the
methodology employed.
Modulation of immune synovitis by antiinflammatory agents was interesting because of the similarities
of this model to RA. These similarities include the occurrence of acute inflammatory exudates composed
mainly of PMNs (1,5-7); infiltration of synovial tissue
by mononuclear cells, including plasma cells (1,4-7);
immunoglobulin synthesis and production of lymphokines, such as macrophage migration inhibitory factor,
by synovial tissue (7,8,23,24): and localization of antibody and complement in avascular structures of the
joint, such as cartilage (2.3).
The acute phase (PMN exudation) of immune
synovitis was suppressed in the present studies for some
steroidal and nonsteroidal agents. Currently available
antiinflammatory drugs do not interfere with the progressive joint destruction in RA. These agents do control, to varying degrees, aspects of inflammation (swelling, pain, stiffness) that accompany joint destruction,
although clinical assessment of these parameters is less
than quantitative. I n addition to the studies presented
here, investigation of the chronic phase of rabbit synovitis may be of further assistance in defining potentially
more effective antirheumatic agents.
ANTIINFLAMMATORY AGENTS A N D IMMUNE SYNOVITIS
Table 5. Effecis
OJ
Other Nonsieroidal Aniiinflammaiory Agenis on ihe Acuie Response ai 6 Hours in
ihe Rabbit Synovitis Tesi
Group Size
~
Compound*
Experiment
lndomethacin
(25 mg/kg)
I
2
3
4
5
6
Phenylbutazone
(100 mg/kg)
I
2
3
Treated,
Control
Percent Decreas,eof Exudatet
OD
4s
7s
779
68
66
(0.03)$
(0.001)
31 (0.06)
44 (0.03)
0
25 (0.2)
66
6.6
6,6
53
54 (0.05)
56 (0.01)
6.9
6.6
21
(0.2)
1
:
.
Acetylsalicylic acid
(300 mg/kg)
Colchicine
945
1
5,6
2
3
43
7,7
1 (2 mg/kg)
2 (2mg/kg)
(0.5 mg/kg)
(0.1 mg/kg)
6-6
6,6
6.6
6.6
(+)I1
74 (0.002)
(0.3)
64 (0.002)
99
(0.001)
75 (0.008)
62
25
(0.004)
(0.2)
Total PMNs
50 (0.03)
16 (0.2)
47 (0.05)
Percent Decrease
of PMN Histology
Score
(+)lo3
34
32
23
14
35
31 (0.07)
98 (0.001)
85 (0.001)
73 (0.003)
40 (0.03)
,(0.5)$
(0.02)
(0.005)
(0.08)
(0.2)
(0.01
10 (0.5)
25 (0.07
35 (0.01
16 (0.2)
(+)I1
1
(0.4)
(0.3)
100 (0.001)
49 (0.003)
34 (0.03)
26 (0.02)
* Compounds were administered by the ip route 0.5 hour before intraarticular challenge with 0.5 mg BSA.
t See Table 3
for explanation.
$ p.
$ (+) indicates an increase in group averages vs controls.
ACKNOWLEDGMENTS
The authors thank Mr. David Cook and Mr. Anatol
Plisko for their capable technical assistance.
REFERENCES
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