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Induction of proliferative synovitis in rabbits by intra-articular injection of immune complexes.

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Induction of Proliferative Synovitis in Rabbits by
Intra-articular Injection of Immune Complexes
By ARNOLDJ. RAWSONAND TITO P. TORRALBA
S
of rheumatoid arthritis (RA)
have implicated immunoglobulins in
the genesis of the local inflammation. Rheumatoid factors (RF), IgG, and IgM have
been shown to be present in, and presumably are produced by, synovial plasma ce1ls.l Polymorphonuclear leukocytes
(PMN) of RA joint fluid have cytoplasmic
granules,Z which contain IgG, IgM3, and,
in many cases, BIG g l ~ b u l i n .Moreover,
~
joint fluid of RA has a relatively decreased
content of several complement ( C ’ ) components when compared to paired serum
samples, and to joint fluid from most other
forms of joint inflammation5j6;material containing IgG or IgM with C’, and IgG with
RF, has been demonstrated in synovial tissues.’ Even patients with very low levels
of serum immunoglobulins may develop arthritis in which immunoglobulins are present in the joint fluid.*
Although proliferative synovitis has been
produced experimentally in hyperimmune
animals by the intra-articular injection of
specific antigen,O>l2there is no convincing
evidence for a hyperimmune state in patients with RA. On the contrary, the observations seem to be consistent with the
reaction of immunoglobulins within the
joint space itself.
The purpose of the present study is to
determine if immunoglobulin reactions localized to the joint space of nonimmunized
experimental animals reproduce the anatomic lesions of RA. Various materials were
injected into the knee joints of normal rab-
bits, and the resulting tissue changes were
observed.
From the Department of Pathology and the
Arthritis Clinic, Uniuerssity of Pennsylvania School
of Medicine, Philadelphia, Pa.
Supported by RG6218, N I H , USPHS.
ARNOLDJ. RAWSON,M.D.: Associate Professor of
Surgical Pathology; TITOP. TOHRALBA,
M.D.: Fellow, Arthritis Section; both of the University of
Pennsylvania School of Medicine.
TUDIES
MATERIALS
AND METHODS
Experimental Animals. Eighteen female albino
rabbits weighing from 3 to 4 Kg. were used. All
injections were given into the knee joints, in most
cases using the contralateral joint as control. The
skin was shaved, sterilized with iodine, and the
joint was entered anteriorly and slightly laterally
with a #22 needle. Sterile technic was observed
throughout. (The experimental plan is summarized
in Table 1; 11 of the 18 animals were injected only
once in acute experiments.) Injections were given
twice weekly for the duration of the experiment.
Knee joints were aspirated occasionally during the
course of the experiment. Animals were sacrificed
with intravenous toxital, 2 ml. At Autopsy, joint
fluid and all synovial membranes were removed
from the knee joints.
Reagents. Egg albumin (EA) 5X crystalline
(Nutritional Biochemicals Corp.) was titrated
against rabbit antiovalbumin (Nutritional Biochemicals Corp.). It was found that equivalence required the use of a 0.025 per cent solution of
antigen. One-half ml. of antigen solution, onehalf ml. of antiserum, and one-half ml. of fresh
autologous serum (FS) as a source of C’ constituted
the injection mixture for this system. The components were injected immediately after mixing.
Pneumococcus type 111 pofysaccharide (PN) and
its specific rabbit antiserum were obtained through
the courtesy of Dr. Gerald Schiffman. The antiser i m (lot 699-17) contained 1.5 mg. antibody N
per ml. The haptene, used a t equivalence, was at a
concentration of 400 pg per ml. One-half ml. each
of antiserum and haptene solution constituted the
injection mixture. FS was not added because C‘
activity was present in the antiserum.
The animals which were hyperiinrnnnizecl were
given 1 ml. of pneumococcus type I11 vaccine
(lot A66) intravenously 4 days a week for 2
weeks, following which 0.5 ml. of haptene solution
44
ARTHRITIS
AND RHEUMATISM,VOL. 10, No. 1 ( FEBHUARY,
1967)
45
INDUCTION OF PROLIFERATIVE SYNOVITIS I N RABBmS
Table 1.-Summaru
Rabbit
Number
10
13
2
8
9
11
14
19
7
18
Left Knee
Injection
AGG-serum
PN-anti P N
PN-anti P N
PN-anti P N
human albumin
AGG-serum
PN-anti PN-RF
AGG*
PN-anti P N
AGG-serum
4
PN-anti P N
3
EA-anti E A
1
12
16
11
EA-anti EA
AGG-saline
PN-anti P N
PN-anti P N
PN
PN
5
6
of Procedure
and Results*
R:ght Knee
Lesions
PMN
PMN-G
PMN-G, S P
PMN-G, S P
N
PMN-G. S P
PMN-G, S P
see text
PMN-G, SP, GC
PMN-G, S P
see text
PMN-G, S P
VIL, PC, F
PMN-G, S P
VIL, PC, F
N
see text
N
N
PMN-G, SP, VAS
PMN-G, SP, VAS
Injection
Lesions
AGG-saline
AGG-serum
serum
PN
human albumin
AGG-RF
PN-anti P N
AGG*-serum
serum
AGG-RF
serum
PMN
PMN
N
N
N
PMN-G, S P
PMN-G, S P
see text
N
PMN-G, S P
VIL, PC. F
N
serum
N
serum
AGG-RF
saline
saline
saline
saline
N
see text
N
N
N
N
General
injected 1 wk. only
zymosan
nitrogen mustard
nitrogen mustard
hyperimmunized
hyperimmunized
Total
Injections
Sacrificed
1
1
1
1
1
1
1
1
2
4
8 hrs.
8 hrs.
3 days
3 days
3 days
3 days
3 days
3 days
5 days
2 wks.
8
4 wks.
12
6 wks.
2
1
1
1
2
2
6 wks.
8 hrs.
3 days
3 days
5 days
5 days
*Except f o r serum or saline, the quantities of which were equal on each side, 0.5 ml. of each reagent was injected.
Serum was fresh, isologous or autologous, each knee receiving the same a s its control. AGG* is labeled with fluorescein isothiocyanate. PMN-G = PMN with cytoplasmic granules: SP = synovial proliferation ; GC = giant cells ; VIL
= villi ; PC = plasma cells : F = follicles : VAS = vasculitis ; N = normal.
was given in one knee and 0.5 ml. saline in the
other.
As a source of rheumatoid factor (RF), serum
from a patient with rheumatoid arthritis was
employed. This serum was stored for 1 year, at
the end of which time C' activity could not be
demonstrated by the sensitized erythrocyte test.
The titer of R F immediately before use was
15120 (latex fixation Difco F 11). One-half ml.
of serum was used for each injection.
Aggregated IgG (AGG) was prepared by heating human gamma globulin, fraction I1 (Pentex,
Inc.) in 1 per cent solution in borate buffered
saline (pH 8.0) at 65 C. for 20 minutes. One-half
ml. was used for each injection.
Controls. The knee opposite to that receiving
the complete immunologic mixture received either
serum alone or antigen alone in the acute experiments. These reagents were made up to comparable volume with isotonic sodium chloride solution.
In the longer experiments, antigen controls were
omitted to avoid sensitization. In no instance was
a lesion seen in the control knee.
Decomplementation. Boiled zymosan in isotonic
sodium chloride solution (15 mg. per ml.) was
administered as follows: At -45 minutes: 1.0 ml.
intravenously (I.V.) and 1.5 ml. intraperitoneally
(1.P.). At -15 minutes: 1.0 ml. I.V., 1.5 ml. I.P.
The same dosages were repeated at +1 hour,
+2 hours, +4 hours, +6 hours. Blood was drawn
at +45 minutes, and again at $4 hours. Activity
of B,, globulin (measured by Dr. Fletcher Taylor)
was 50 per cent of normal in both specimens.
The assay was performed by selective destruction
of B,, globulin activity with hydrazine, after
prior neutralization of C', esterase with anti-C',
serum.
Depletion of P M N . Nitrogen mustard was given
intravenously (1.75 mg. per Kg.), and the blood
leukocytes were examined daily thereafter. When
the PMN count reached zero (1-2 days) the
experiment was begun. The animals were maintained throughout on 125 mg. streptomycin and
150,000 units penicillin daily.
Histologic Technic. Synovial membranes were
fixed in neutral buffered formalin, embedded in
paraffin, sectioned, and stained with hematoxylin
and eosin. Joint fluids were centrifuged, and the
sediment washed 3 times in isotonic sodium chloride solution, and fixed 10 minutes in acetone.
Fhosescence Studies. Fluorescein-labeled goat
antirabbit globulin serum (Hyland) was employed
for study of joint fluid cells. Specificity was
controlled by inhibition with nonfluorescent antiserum.
AGG was labeled with fluorescein isothiocyanate
(1 mg. per 40 mg. protein). The mixture was
kept at pH 9.2 to 9.5 for 1 hour, stored at 4 C.
overnight, and taken off sephadex G50 with borate
buffer pH 8.2 (AGG").
46
RAWSOh' AND TORRALBA
Fig. 1A.-Rabbit 13 (left knee). Joint fluid cells stained with fluorescein-labeled
goat anti-rabbit IgG serum. Note fluorescent intracytoplasmic granules. x 750. Duration
of experiment: 8 hours.
in more PMN, but far fewer than in the
RESULTS
An acute inflammatory reaction was well nondecomplemented animals. The synovial
established eight hours after the intra- membranes, following both injection mixarticular injection of PN-anti PN, EA-anti tures, showed a few PMN within or near
EA, AGG-RF, AGG-FS, or AGG. The joint venules and a few at the synovial surface.
space contained turbid fluid and many Other inflammatory features were lacking.
PMN. Intracytoplasmic granules were not
Three days after injection of either AGGprominent following the injection of AGG FS or AGG-RF into intact animals there
or AGG-FS, but granules were numerous was minimal joint fluid with rare PMN
after injection of other systems and were containing a few granules. The synovial
specifically stained with goat antirabbit membranes showed a slight proliferative
gamma globulin labeled with fluorescein reaction of the synovial cells. An animal
(Fig. 1A). The synovial membrane was receiving fluorescein-labeled AGG in one
edematous, the synovial cells showed nu- joint and labeled AGC together with FS in
clear enlargement and rounding, and nu- the other showed only traces of fluorescence
merous PMN were clustered along the in the synovial membrane of both joints
after three days.
synovial surface (Fig. 1B ).
Three days after injection of PN-anti PN,
The acute reaction was somewhat modified in the zymosan-treated animal. The however, there was prominent proliferation
injection of AGG-FS was followed by mini- of the synovial cells. PMN were not seen
mal joint fluid and only a rare PMN in among the proliferated cells, but were coneight hours. Injection of AGG-RF resulted centrated along the synovial surface. Small
INDUCTION OF PROLIFERATIVE SYNOVITIS IN RABBITS
47
Fig. 1B.-Rabbit 13 (left knee). Synovial membrane. Note clustering of PMN on
surface. H. and E., x 280. Duration of experiment: 8 hours.
vessels of the synovia showed no evidence
of vasculitis (Fig. 2).
Five days after the initial injection of
PN-anti PN, there was even more marked
proliferation of synovial cells. Mitotic figures were numerous, and many multinucleated giant cells were seen (Fig. 3 ) .
PMN containing granules were present in
the synovial cavity, but were not seen in
the synovial membrane.
Two weeks after the initial injection of
AGG-FS there was moderate proliferation
of the synovial cells (Fig. 4A) with scattered clumps of fibrinous material on the
surface. A few small inconspicuous villi
were seen, and rare plasma cells were in
the synovia. PMN containing granules were
present in the joint space, but not in the synovial membrane. After four injections (in
two weeks) of AGG-RF there was marked
synovial cell proliferation. Villi were large
and prominent. Throughout the proliferat-
ing area were numerous plasma cells and
early lymphoid follicles (Fig. 4 B ) . Multinucleated giant cells were also present.
PMN with granules were numerous in
the synovial fluid, but absent from the
membrane.
After 8 (in 4 weeks) injections with
PN-anti PN, and after 12 (in 6 weeks)
injections with EA-anti EA, there was
gross enlargement of the joint with thickening of the soft tissues and granulomatous
proliferation appearing microscopically as a
pannus covering the cartilage (Fig. 5A).
There were very large, prominent villi containing lymphoid follicles devoid of germinal centers, closely resembling those seen
in human RA (Fig. 5B). The t'issues were
heavily infiltrated by plasma cells. Although
PMN were numerous in the joint space,
they were absent from the synovial tissues.
Animals made agranulocytic with nitrogen mustard received injections of PN-anti
48
RAWSON AND TORRALBA
Fig. 2.-Rabbit 2 (left knee). Synovial membrane. Note marked proliferation of
synovial cells, with surface clustering of PMN, but no inflammation of venule at lower
left. H. and E., x 280. Duration of experiment: 3 days.
Fig. 3.-Rabbit
7 (left knee). Synovial membrane. Note marked synovial cell
proliferation and multinucleated giant cells. H. and E., x 280. Duration of experiment: 5 days.
INDUCTION OF PROLIFERATIVE SYNOVITIS IN RABBITS
Fig. 4A.-Rabbit 18 (right knee). Synovial membrane after injection of AGG-serum.
Synovial cell proliferation. H. and E., x 280. Uuration of experiment: 2 weeks
(compare with Fig. 4B).
Fig. 4B.-Rabbit 18 (right knee). Synovial membrane after injection of AGG-RF.
Villous synovitis with formation of follicles composed of plasma cells and lymphocytes.
H. and E., x 280. Duration of experiment: 2 weeks (compare with Fig. 4 A ) .
49
50
RAWSON AND TORRALBA
Fig. 5A.-Rabbit 4. Left knee (left) syiiovial membrane after injection of PN-anti
PN. Shows marked thickening of synovium with some pannus formation. (Duration of
experiment: 4 weeks).
Fig. 5B.-Rabbit 4 (left knee) synovial membrane after injection of PN-anti PN.
There is villous synovitis with lymphoid follicles and heavy plasma cell infiltrate. H.
and E., x 280. Duration of experiment: 4 weeks.
INDUCTION OF PROLIFERATIVE SYNOVITIS IN RABBITS
51
response; multiple injections induced a recurrent acute response and produced the
typical anatomic lesions of RA. The injected
material included a protein antigen, a polysaccharide haptene, and RF-AGG. AGG
injections alone produced an arthritis, but
changes were less typical of RA.
Constant presence of the reactants appeared to be necessary, as there was a
tendency, at least in the early stages, for
the process to regress if injections were
discontinued. Labeled AGG was almost
completely removed from the joint cavity
within three days.
Although treatment with zymosan did
not remove all the Blc globulin, infiltration
of PMN into the injected joint was reduced.
Interpretation of this finding is uncertain,
since this treatment also may depress the
level of circulating leukocytes and, unfortunately, blood counts were not obtained in
these experiments. In view of the demonstration of chemotactic properties of C’oC6,l0J1
it is likely that the C’ system may
be of importance in the initial attraction
of PMN to the joint.
The failure of synovial proliferation to
develop in animals made leukopenic with
nitrogen mustard suggests that the lesions
are mediated by PMN2 rather than by a
direct injury to the synovial cells themselves. The ability of streptolysin S to induce acute and chronic arthritis13 suggests
that tissue damage may be mediated by
substances released from damaged lysosomes. Here the postulated lysosomal damage may be from PMN or from synovial
lining cells.
The tissue changes appearing in the hyperimmune animal are very different from
those appearing in the nonhyperimmune
animal and involve necrotizing vasculitis,
a process not seen in the joints of RA. It
DISCUSSION
is tempting to speculate that a hyperThese experiments demonstrate that sin- immune state may have developed in those
gle injections of various antigen-antibody rheumatoid patients whose course is comsystems into normal rabbit joints are cap- plicated by vasculitis.
able of inducing an acute inflammatory
PN into the knee joints according to the
same schedule as that of the intact animals. Three days after the initial injection
the animals were sacraced: PMN were
absent from both the joint cavity and synovial tissues, no significant joint fluid was
present, and the synovial cells showed no
evidence of proliferation. The tissues were
indistinguishable from those of the normal
control knee.
The joints made hyperimmune to type 111
pneumococcus, of animals which received
intra-articular injections of PN over a 5 day
period, contained fluid and numerous PMN
with intracytoplasmic granules, which
showed specific staining with fluoresceinlabeled goat antirabbit globulin serum.
There was marked synovial cell proliferation comparable with that noted after the
injection of PN-anti PN into the joints of
normal animals over a 5 day period. The
hyperimmune animals differed from the
nonhyperimmune ones, however, in that in
the former PMN were present in large
numbers throughout the synovial membrane instead of being limited to the synovial surface. In addition, there was severe
vasculitis, with necrosis of venular walls,
and concentration of PMN in these areas.
Extensive fibrinoid necrosis of synovial
membrane was also seen.
In rabbit No. 14, an attempt was made
to determine the effect of R F on the biologic activity of an antigen-antibody system.
No significant alteration in response could
be seen when RF was added to the freshly
mixed reagents just prior to injection.
An animal which received intra-articular
human serum albumin (0.5 per cent in
saline), aggregated by heating to 80 C.,
showed no inflammatory response after 3
days.
52
RAWSON AND TORRALBA
SUMMARY
Injection of various immune systems, including complexes of rheumatoid factoraggregated gamma globulin, into normal rabbit joints induces t h e anatomic lesions
similar to those of rheumatoid arthritis when introduced into normal rabbit joints.
The development of these lesions seems to depend upon t h e continued presence of
t h e reactants and the availability of polymorphonuclear leukocytes. Injections into
hyperimmune animals induced vasculitis in addition to an inflammatory reaction.
SUMMARIO
IN INTERLINGUA
Varie systemas immun-incluse
complexos de factor rheuinatoide con aggregate
globulina gamma-induce
le lesiones anatomic de arthritis rheumatoide quando illos
es introducite ad in le articulationes de conilios normal.
Le disveloppamento del lesiones pare depender del presentia continue del reactantes e del disponibilitate de leucocytos polymorphonucleari. Injectiones in animales
hyperimmun induceva vasculitis a parte le reaction inflammatori.
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2. Hollander. J. L.,McCarty, Jr., D. J., Astorga,
G., and Castro-Murillo, C.: Studies on the
pathogenesis of rheumatoid joint inflammation. I. The “R. A.” cell and a working
hypothesis. Ann. Int. Med. 62:271, 1965.
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J. L.: Studies on the pathogenesis of rheumatoid joint inflammation. 11. Intracytoplasmic particulate complexes in rheumatoid
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5. Pekin, T. J.. and Zvaifler, N. J.: Hemolytic
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8. Collins, J. R., and Ellis, D. S.: Agammaglobulinemia, malabsorption, and rheumatoid-like
arthritis. Amer. J. Med. 39:476, 1965.
9. Klinge, F.: Der Rheumatismus: Die experimentelle serumhyperergie. Ergebn. Allg.
Path. 27:245, 1933.
10. Ward, P. A., and Cochrane, C. G.: Bound
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11. Ward, P. A., Cochrane, C . G., and MiillerEberhard, H. J.: The role of serum complement in chemotaxis of leukocytes in uitro.
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12. Glynn, L. E.: Arthropathy in man and animals.
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13. Weissmann, G., Becher, B. S., Wiedermann,
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