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Experimental viral arthritis induced with herpes simplex.

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Experimental Viral Arthritis Induced With Herpes Simplex
Francis W. Webb, Rodney Bluestone, Leonard S . Goldberg, Steven D.
Douglas and Carl M. Pearson
A chronic erosive arthritis was produced in rabbits by a single intraarticular injection of herpes simplex virus. Three months after injection, a
moderate or severe arthritis was present in the injected knees of all 10
animals; mild or moderate changes were present in 9 of the contralateral
(noninjected) knee joints. The histologic changes resemble those of
human rheumatoid arthritis. Thus, under certain circumstances, a single
exposure to herpes simplex virus appears to provoke chronic arthritis.
Although the etiology of rheumatoid arthritis
(RA) is unknown, it seems possible that an infectious agent initiates the disease (1-3). However, no specific bacterial organism has consistently been recovered from human rheumatoid
synovial tissue. Conceivably, many of the histopathologic and immunologic features of RA
From the Departments of Medicine Veterans Administration Wadsworth Hospital Center, University of California at Los Angeles Center for the Health Sciences and the
Mount Sinai School of Medicine, Los Angeles, Calif and
New York, NY.
Supported in part by grants from the Southern California
Chapter of the Arthritis Foundation, the US Public Health
Service (GM15759) and the Veterans Administration.
FRANCIS w. WEBB, MB, MRCP: Department of Rheumatology and PM & R, Norfolk-Norwirh Hospital, Norfolk,
England; RODNEY BLUESTONE, MB, MRCP: Assistant Professor of Medicine, University of California at Los Angeles
School of Medicine, and Chief of Rheumatology. Veterans
Administration Wadsworth Hospital Center, Wilshire and
Sawtelle Boulevards, Los Angeles, Calif 90073; LEONARD s.
GOLDBERG, MD: Assoriate Professor of Medicine, University
of California at Los Angeles School of Medicine, Center for
Health Sciences, Los Angeles, Calif; STEVEN D. DOUGLAS,
MD: Assistant Professor of Medirine. Department of Medicine, Laboratory of Cellular and Subcellular Immunology,
Mount Sinai School of Medicine, New York, NY; CARL M.
PEARSON, MD: Professor of Medicine, University of California at Los Angeles School of Medicine, and Director, Rheumatology Division, University of California at Los Angeles,
Reprint requests should be addressed to: Dr. Rodney
Submitted for publication June 27, 1972; accepted August 31, 1972.
could be related to an episodic or chronic viral
infection (4). Indeed, several clinical situations
have been described in which acute transient
synovitis is associated with viraemia and in
which viral material can be recovered from the
joint space (5,6).
Numerous animal models of acute and
chronic bacterial arthritis have been studied.
However, little information is available on the
production of an experimental chronic viral arthritis. In the present study, herpes simplex
virus (HSV) was chosen as a possible arthritogenic agent for several reasons. First, there is
some evidence that patients with RA may develop humoral immunologic tolerance to the
virus (7), although the validity of this observation has recently been questioned (8). Further,
recent evidence suggests that the virus can exist
in latent form within nerve tissue of experimental animals (9); by doing so it defies isolation and identification by conventional technics.
Such a mechanism might explain the mainly
negative culture studies on synovial membrane
in RA (1 0 ) . Secondly, herpes virus incorporates
host antigen into its envelope and thus might
evoke an autoimmune response similar to that
typifying human connective tissue disease (1 1).
Indeed, a potential biologic role for rheumatoid
factor might be to enhance the neutralization of
herpes virus-antibody (I&) complexes (12).
Lastly, the virus is ubiquitous and relatively
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
Fig 1(top). Normal synovium. One layer of flattened synoviocytes and subsynovial fat ( x 300).
Fig 2 (bottom). Mild synovitis (+), showing a double layer of somewhat rounded lining cells
( x 85).
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
safe to handle in the laboratory.
This report describes a chronic proliferative
synovitis and an erosive arthritis which is induced in rabbits by HSV.
Eighteen young, random-bred, female New Zealand
white rabbits weighing approximately 3 kg were used
(ABC Aviary and Rabbitry. Pomona, Calif).
Preparation of Herpes Simplex Virus
T h e HSV hominis (H, strain)* was grown on glass in
monolayers of RK 13 rabbit kidney cells (RKC). T h e cells
were gently srraped from the glass, centrifuged at 1200s for
5 minutes and the cell pellet containing approximately 5 x
10* rells was resuspended in 0.5 ml of minimal essential medium (MEM) containing 5% fetal calf serum, penicillin,
200 units/ml, and streptomycin, 200 Cg/ml. T h e cells were
then disrupted by sonication at 4" C (Branson S-75 sonifier
microtip, tap 7.2.5 amps for 1 minute) centrifuged at l0OOg
for 10 minutes to remove RK 13 cell debris and stored undiluted at -70" C until needed. Uninferted parallel cultures of
RKC were similarly sonicated. T h e infected sonicate was
diluted to actually contain 1 x lo6plaque forming units of
virus per ml prior to use (13). At this concentration of virus
the protein content of both infected (HSV) and uninfected
(RKC) sonicates were approximately 50 rg/ml when measured by Lowry's method (14).
Subsequently, the RK 13 cell line was found to he contaminated with Mycoplasma laidlawii. This is a common
saprophytic mycoplasma which can he present in sera used
for cell cultures. However, subsequent experiments revealed
that the injection of rontrol animals with HSV-free. contaminated RK 13 sonicates failed to produce arthritis in 7 of
8 animals.
Knee Joint Injections
Using continuous intravenous thiopental for general
anesthesia, the fur was clipped away anteriorally over the
knee and the skin cleaned with iodine. With the knee flexed,
a 23-gauge needle was inserted through the medial edge of
the patellar ligament from the front, using an aseptir technic. One ml of HSV or RKC was then in,jected with particular attention being paid to slight medial and lateral distention of the joint capsule, indirating a truly intraarticular
Preparation of Emulsions and IntraLymph Node Immunization
Heavy mineral oil (Squibb) with 5% Arlacel (Atlas
Chemical Inc, Wilmington, Del) as an emulsifying agent
was shaken thoroughly with finely ground, dead, dried human tubercle bacilli of strains C , DT and PN.* Equal volumes of HSV or RKC sonicate (approximately 50 gg/ml
protein rontent) were added slowly with agitation to the oil
suspension and emulsification completed by repeatedly
passing the mixture through a 23- or 26-gauge needle to
produce a water-in-oil emulsion. T h e concentration of the
reagents in the emulsions used for immunization were: tubercle bacilli, 1.0 mg/ml; HSV, 0.5 x lo6 plaque forming
units per ml; RKC (virus-free sonicate). 25 r g protein per
ml. Immunizations were made under general anesthesia
with 0.1 ml of an emulsion into each popliteal lymph node
through a 30-gauge needle.
Experimental Procedure
Eighteen rabbits were divided into two groups.
Group 1 animals, 10 in number, were injected with HSV
into the left knee joint. From pilot studies it was anticipated
that a n acute viral synovitis would he induced and that the
production of a chronic synovitis might depend on further
systemic challenge with the viral antigen. Therefore, 2
weeks after intraarticular in.jection, 4 of the Group I animals were immunized with HSV emulsion administered
into the popliteal nodes.
Group 2 animals, 8 in number, were injected with RKC
into the left knee joint. T w o weeks later, 6 of this group
were immunized with RKC emulsion injected into the popliteal nodes.
All animals were sacrificed 3 months after receiving the
intraarticular injections.
Preparation and Examination of
Histologic Specimens
After sacrifice, both knee joints of each animal were dissected, excised in toto, partly opened anteriorly to obtain
small pieces of synovium for electron microscopic examination and then fixed in buffered formalin and decalcified
in freshly mixed equal volumes of 30% formic acid and 2 ,I1
sodium formate. Three large sections of each joint providing
composite views of the articular structures were cut as follows: a) coronal section through the femoral condyles; h)
sagittal section through the detached patella and c) sagittal
section through the artirular region of the tibia.
These sections gave ample synovial membrane, articular
cartilage, subchondral bone and ligamentous insertions for
examination. Sections were stained with haematoxylin and
eosin and examined independently by 2 observers. A system
* Kindly provided by the Central Veterinary Laboratory.
* Kindly prepared and provided by Dr. J a c k G . Stevens.
Weybridge, England.
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
WE00 E l AL
Fig 3 (top). Moderate synovitis ( + +). multiple layers of heaped-up synoviocytes with
surface pseudopods. subsynovial edema, infiltrating mononuclear cells and some
fibroblastic proliferation ( x 60). Fig 4 (bottom). Moderately severe synovitis (+ +).
characterized by a synovial lymphoid aggregation ( x 240).
of grading was used in a n attempt to quantitate histologic
changes, defined as follows:
0 = Normal synovium (Figure I ) : one to two layers
of flattened synovial cells with subsynovial fat abutting
onto membrane.
+ = Mild synovitis (Figure 2): two to three layers
flattened or somewhat rounded synovial cells; scanty
mononuclear infiltrate.
+ + = Moderate synovitis (Figure 3): three to four
or more layers of rounded cells; definite synovial hyperplasia; many mononuclear cells.
Arthritis and Rheumatism, Vol. 16. No. 2 (March-April 1973)
Fig 5. S e v e r e synovitis ( + + + +) exemplified by invasive synovial m e m b r a n e ( p a n n u s ) e x t e n d i n g b o t h onto t h e s u r f a c e of a r t i c u l a r cartilage a n d into cartilage a n d s u b c h o n d r a l b o n e ( x 300).
+++ = Moderately severe synovitis (Figure 4):
moderate synovitis with lymphoid aggregation.
+ ++ + = Severe synovitis (Figure 5): moderately
severe synovitis with marginal bony erosions and/or
periostitis (Figure 6).
Representative pieces of freshly obtained synovial membranes were fixed in 2% glutaraldehyde in 0.1 M sodium
cacodylate and postfixed in 1% aqueous uranyl acetate, rapidly dehydrated and embedded in Epon. Thin sections were
prepared on an LKB Ultrotome 111 and were doubly
stained with uranyl acetate and lead citate (15) and examined in a Siemens 101 Electron Microscope. Multiple thin
sections were examined in detail from 5 animals.
Skin Tests
Intradermal skin tests were performed before, and at two
weekly intervals after intraarticular injection using 0.1 ml
doses of a solution of HSV containing 1.0 x lo6 plaque
forming units per ml, and/or sterile RKC containing SO pg
protein per ml. Additional skin tests were performed with
PPD, 100 pg/ml. Skin tests were regarded as positive if an
easily palpable induration of at least 10 mm diameter with
an increase of double skin thickness of greater than 0.3 mm
was detected at 48 hours (Quirk test dial Caliper Gauge
(Type A02T) Krnplin, Hessen, West Germany).
Serologic Studies
Rabbit sera were obtained from each animal before intraarticular injection and at 2 weekly intervals thereafter.
Rheumatoid factor was looked for in heat-inactivated
sera using sheep red cells sensitized with rabbit IgG (16)
and latex particles coated with aggregated human I$,
Antinuclear antibody was determined by indirect immunofluorescence ( I S ) using rabbit huffy coat white cells and
fluoroscein-conjugated goat antiserum to rabbit immunoglobulin (Kallestad Laboratories. Minneapolis. Minn). Antibodies to D N A were measured by agarose immunodiffusion (19) and by a DNA-binding assay (20) using calf
thymus D N A (Worthington Biochemicals, Freehold, NJ)
labelled with Actinomycin D (H’) (Schwartz/Mann, Orangeburg, NY). Positive and negative human serum controls were included in the assays.
Antibodies to HSV were quantitated by complement fix-
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
Fig 6. Femoral shaft adjacent to knee joint showing intense periostitis with active
fibroplasia and almost complete lysis of surface cortical bone. Quadriceps muscle
(above) is unaffected ( x 40).
ation (21). An aliquot of HSV used for injection was heat
inactivated and served as antigen. Complement source was
lyophilized guinea pig complement (Hyland Laboratories,
Costa Mesa, Calif). .4 known high-titer positive rabbit serum was used as a control.
some-like organelles, and the other was a cell
characterized by the presence of well-developed,
rough-surfaced endoplasmic reticulum. Occasional plasma cells were observed. No virus-like
particles were present in the thin sections examined.
Group 2 Animals (Table 2). One animal
a moderately severe, bilateral synGroup 1 animals (Figures I to 6 and
animals in this group revealed
Table I ). Moderate (++) to severe (++ + +)
whether or not they had
synovitis was found in the injected'joints of all
the animals in this virus-injected group. Mild
(+) to moderate (++) changes were present in
all but one of their contralateral, noninjected
knee joints. No difference in severity of synovitis was detected between those animals who
received an intranodal immunization with HSV
and those who did not.
Electron microscopy revealed that the synovium was comprised of the two general types of
cells described recently from other laboratories (22-24). One, a phagocytic cell, contained numerous single membrane-bound, lyso246
been subsequently immunized by the intranodal
administration of RKC emulsion.
Immune Response
Skin tests. Skin tests to HSV, RKC: and
PPD remained negative in all Group 1 animals
who had not been subsequently immunized by
the intranodal injection of HSV emulsion. Of
the 4 in the group who were immunized later, 2
gave positive tests to HSV, and all 4 gave positive tests to PPD. None reacted to intradermally
administered RKC.
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
Table 1. Histologic Gradings of Both Knees:
Group 1Animals, HSV* Injected into Left Knee
Table 2. Histologic Gradings of Both Knees: Group
2 Animals, RKC* Injected into Left Knee
Histologic severity
of synovitis
in both knees1
"1 x 10 plaque forming units of HSV in 1 ml of
RKC culture medium
?Rabbits 7 through 10 received intralymph node
immunization with HSV in adjuvant 2 weeks after
intraarticular injection.
ISee text
Skin tests to RKC: and PPD in Group 2 animals who had been subsequently immunized
with R K C in adjuvant became uniformly and
strongly positive. These animals were not tested
with the HSV preparation in order to minimise
the risk of inadvertent herpes infection among
the control group.
Herpes A n t i b o d i e s . All a n i m a l s in
Group 1 showed a rising titer of antiherpes antibody (range: 1 :2 to 1 :256, mean maximum titer: 1 :32). No positive antibody titers were detected in Group 2 animals.
Serology. Tests for serum rheumatoid factor, antinuclear antibody and anti-DNA antibody were uniformly negative in both groups.
This experiment has demonstrated that a
ubiquitous infective organism-herpes simplex
virus-can evoke a chronic erosive synovitis in
Histologic severity
of synovitis
i n both knees1
*One milliliter of uninfected RKC culture medium
containing 50 pg protein
?Rabbits 3 through 8 received intralymph node
immunization with RKC in adjuvant 2 weeks after
intraarticular injection.
$See text
rabbits. Although the more severe changes were
seen where the virus was introduced directly
into the joint cavity, the development of mild or
moderate changes in the noninjected joint suggests that a polyarthritis, possibly related to a
viremia, might have been induced. Subsequent
reexposure to viral antigen was not necessary to
produce chronic synovitis; the inflammatory
changes were equally severe whether or not the
animal had been immunized with HSV after intraarticular injection. It is most unlikely that
the mycoplasma contaminant within the RK 13
cell line was alone responsible for the arthritis,
since a chronic synovitis was seen in only 1 of 8
animals given intraarticular R K C without herpes virus. A synergistic effect between HSV and
mycoplasma in the virus-injected animals is
conceivable but seems equally unlikely. Nevertheless, the role of HSV as an adjuvant for
mycoplasmal infection cannot be totally excluded, and experiments are in progress to further clarify this point.
T h e nature of this induced experimental arthritis resembles human RA in many ways.
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
Synovial cell hyperplasia, lymphocytic infil- duced anergy. Anergy accompanying viral intration with follicle formation and subsynovial fections is well-recognized in animals and
new vessel formation was prominent. In addi- man (26). Interestingly, current evidence sugtion, certain rabbits demonstrated frank erosion gests that cell-mediated immunity plays a
of articular cartilage and reactive periostitis at greater role in host defense against herpes virus
the site of tendon insertions associated with than does humoral antibody (27). However, at
bony destruction. All these changes were appar- present, our numbers of experimental animals
ent by 3 months, and further work is needed to are too small to correlate anergy (as shown by
elucidate the rate of evolution and the chronic- negative skin tests) with severity of synovitis.
O n e control animal (Group 2) developed a
ity of this pathologic process.
Experience from some acute human viral moderately severe bilateral synovitis which is
syndromes, together with the observations out- difficult to explain. It seems unlikely that this
lined here, suggest that the synovial membrane could have been a bilateral mycoplasmal arthrimacrophages may act as filters, sequestrating tis since none of the other 7 animals in this
circulating viral particles (5, 6, 25). It would be group showed this effect. A direct irritant or imof great interest to know if the development of a mune response to the RK 13 protein likewise
chronic arthritis is dependent on viral persis- appears doubtful since the synovitis was present
tence. Conceivably. the herpes virus might have in both the injected and noninjected knee. In
initiated an inflammatory reaction within the fact, all those animals in Group 2 who were
synovial membrane which became self-perpetu- subsequently immunized were at risk of develating in the absence of persistent infection. T h e oping a unilateral nonspecific immune arthritis
existence of a similar mechanism in man would as described by Dumonde and Glynn (28). In
raise interesting implications, particularly in the experimental induction of a n immune
regard to the pathogenesis of RA. Although arthritis, the intraarticular injection of approxelectron microscopic examination of selected imately 100 pg of protein antigen usually
pieces of synovial tissue from some of these ani- suffices to produce chronic synovitis if admals failed to reveal typical viral particles, the ministered after systemic immunization with
limited sampling does not exclude their pres- that protein (29). Conversely, small amounts of
ence. Moreover, the latency of herpes virus is the protein antigen within the joint will usually
well-recognized, and positive identification of evoke a similar arthritis when the intraarticular
persistent infection would be most important injection precedes systemic immunization (30).
and requires specialized virologic technics (9). Under both circumstances, intraarticular perSuch a study is currently underway in our labo- sistence of antigen appears to be important. Although our control animals were injected inratories.
T h e experiment reported here revealed lim- traarticularly with 50 pg of protein contained
ited data on the possible role of the immune re- within the HSV-free RKC preparation, 5 of 6
sponse to the virus and its relationship to the animals failed to show an ipsilateral chronic
subsequent synovitis. Humoral antibody to her- immune synovitis following systemic immunipes was apparent in all those animals injected zation.
It is also possible that the chronic synovitis
with live virus, but autoantibodies of the type
characterizing human connective tissues disease described in this experiment is, by chance, spewere not found. However, the predominantly cific for both virus and host. In this regard, the
negative skin responses to virus and kidney cell effects of other common viruses in laboratory
culture medium in animals injected with live animals would be of considerable interest. Notvirus (and subsequently immunized with HSV withstanding, more detailed study of herpes
in adjuvant) suggests some degree of viral-in- simplex arthritis in rabbits may serve to shed
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
important light u p o n some of the pathogenetic
mechanisms operative i n h u m a n arthritis.
The authors gratefully acknowledge the skilled technical
assistance of Mrs. Robin Yeaton, Mrs. Barbara Vande
Sande and Miss Jo Ellen Cunningham. Dr. J. G . Stevens
provided much helpful discussion.
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